N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-A][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide, a dual modulator of chemokine receptor activity, crystalline forms and processes

ABSTRACT

The present invention provides a novel antagonist: N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide: 
                         
or a pharmaceutically acceptable salt, solvate or prodrug, thereof, having unexpected dual CCR-2 and CCR-5 receptor activity. Crystalline forms, metabolites, pharmaceutical compositions containing the same and methods of using the same as agents for the treatment of inflammatory diseases, allergic, autoimmune, metabolic, cancer and/or cardiovascular diseases are also disclosed. The present disclosure also provides processes for preparing compounds of Formula (I) as provided herein, including N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide. Compounds that are useful intermediates of the process are also provided herein.

This application claims the benefit of U.S. Provisional Application No.61/250,978, filed Oct. 13, 2009, incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention providesN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,or a pharmaceutically acceptable salt, solvate or prodrug, thereof,having unexpected desirable dual activity. Crystalline forms of thepresent invention are also provided. Pharmaceutical compositionscontaining the same and methods of using the same as agents for thetreatment of inflammatory diseases, allergic, autoimmune, metabolic,cancer and/or cardiovascular diseases is also an objective of thisinvention. The present disclosure also provides a process for preparingcompounds of Formula (I), includingN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide:

wherein R¹, R⁸, R⁹, R¹⁰, and

are as described herein. Compounds that are useful intermediates of theprocess are also provided herein. Metabolites of active compounds,pharmaceutical compositions, and use thereof are also provided herein.

BACKGROUND OF THE INVENTION

Chemokines are chemotactic cytokines, of molecular weight 6-15 kDa, thatare released by a wide variety of cells to attract and activate, amongother cell types, macrophages, T and B lymphocytes, eosinophils,basophils and neutrophils (reviewed in: Charo et al., New Eng. J. Med.,354:610-621 (2006); Luster, New Eng. J. Med., 338:436-445 (1998); andRollins, Blood, 90:909-928 (1997)). There are two major classes ofchemokines, CXC and CC, depending on whether the first two cysteines inthe amino acid sequence are separated by a single amino acid (CXC) orare adjacent (CC). The CXC chemokines, such as interleukin-8 (IL-8),neutrophil-activating protein-2 (NAP-2) and melanoma growth stimulatoryactivity protein (MGSA) are chemotactic primarily for neutrophils and Tlymphocytes, whereas the CC chemokines, such as RANTES, MIP-1α, MIP-1β,the monocyte chemotactic proteins (MCP-1, MCP-2, MCP-3, MCP-4, andMCP-5) and the eotaxins (-1 and -2) are chemotactic for, among othercell types, macrophages, T lymphocytes, eosinophils, dendritic cells,and basophils. There also exist the chemokines lymphotactin-1,lymphotactin-2 (both C chemokines), and fractalkine (a CX₃C chemokine)that do not fall into either of the major chemokine subfamilies.

The chemokines bind to specific cell-surface receptors belonging to thefamily of G-protein-coupled seven-transmembrane-domain proteins(reviewed in: Horuk, Trends Pharm. Sci., 15:159-165 (1994)) which aretermed “chemokine receptors.” On binding their cognate ligands,chemokine receptors transduce an intracellular signal though theassociated trimeric G proteins, resulting in, among other responses, arapid increase in intracellular calcium concentration, changes in cellshape, increased expression of cellular adhesion molecules,degranulation, and promotion of cell migration. There are at least tenhuman chemokine receptors that bind or respond to CC chemokines with thefollowing characteristic patterns (reviewed in Zlotnik et al., Immunity,12:121 (2000)): CCR-1 (or “CKR-1” or “CC-CKR-1”) [MIP-1α, MCP-3, MCP-4,RANTES] (Ben-Barruch et al., Cell, 72:415-425 (1993), and Luster, NewEng. J. Med., 338:436-445 (1998)); CCR-2A and CCR-2B (or“CKR-2A”/“CKR-2B” or “CC-CKR-2A”/“CC-CKR-2B”) [MCP-1, MCP-2, MCP-3,MCP-4, MCP-5](Charo et al., Proc. Natl. Acad. Sci. USA, 91:2752-2756(1994), and Luster, New Eng. J. Med., 338:436-445 (1998)); CCR-3 (or“CKR-3” or “CC-CKR-3”) [eotaxin-1, eotaxin-2, RANTES, MCP-3, MCP-4](Combadiere et al., J. Biol. Chem., 270:16491-16494 (1995), and Luster,New Eng. J. Med., 338:436-445 (1998)); CCR-4 (or “CKR-4” or “CC-CKR-4”)[TARC, MDC] (Power et al., J. Biol. Chem., 270:19495-19500 (1995), andLuster, New Eng. J. Med., 338:436-445 (1998)); CCR-5 (or “CKR-5” OR“CC-CKR-5”) [MIP-1α, RANTES, MIP-1β] (Samson et al., Biochemistry,35:3362-3367 (1996)); CCR-6 (or “CKR-6” or “CC-CKR-6”) [LARC] (Baba etal., J. Biol. Chem., 272:14893-14898 (1997)); CCR-7 (or “CKR-7” or“CC-CKR-7”) [ELC] (Yoshie et al., J. Leukoc. Biol., 62:634-644 (1997));CCR-8 (or “CKR-8” or “CC-CKR-8”) [1-309](Napolitano et al., J. Immunol.,157:2759-2763 (1996)); CCR-10 (or “CKR-10” or “CC-CKR-10”) [MCP-1,MCP-3] (Bonini et al., DNA Cell Biol., 16:1249-1256 (1997)); and CCR-11[MCP-1, MCP-2, and MCP-4] (Schweickart et al., J. Biol. Chem., 275:9550(2000)).

In addition to the mammalian chemokine receptors, mammaliancytomegaloviruses, herpesviruses and poxviruses have been shown toexpress, in infected cells, proteins with the binding properties ofchemokine receptors (reviewed in: Wells et al., Curr. Opin. Biotech.,8:741-748 (1997)). Human CC chemokines, such as RANTES and MCP-3, cancause rapid mobilization of calcium via these virally encoded receptors.Receptor expression may be permissive for infection by allowing for thesubversion of normal immune system surveillance and response toinfection. Additionally, human chemokine receptors, such as CXCR4,CCR-2, CCR-3, CCR-5 and CCR-8, can act as co-receptors for the infectionof mammalian cells by microbes as with, for example, the humanimmunodeficiency viruses (HIV).

The chemokines and their cognate receptors have been implicated as beingimportant mediators of inflammatory, infectious, and immunoregulatorydisorders and diseases, including asthma and allergic diseases; as wellas autoimmune pathologies, such as rheumatoid arthritis and multiplesclerosis; and metabolic diseases, such as atherosclerosis and diabetes(reviewed in: Charo et al., New Eng. J. Med., 354:610-621 (2006); Gao,Z. et al., Chem. Rev., 103:3733 (2003); Carter, P. H., Curr. Opin. Chem.Biol., 6:510 (2002); Trivedi et al., Ann. Reports Med. Chem., 35:191(2000); Saunders et al., Drug Disc. Today, 4:80 (1999); Premack et al.,Nature Medicine, 2:1174 (1996)). For example, the chemokine monocytechemoattractant-1 (MCP-1) and its receptor CC Chemokine Receptor 2(CCR-2) play a pivotal role in attracting leukocytes to sites ofinflammation and in subsequently activating these cells. When thechemokine MCP-1 binds to CCR-2, it induces a rapid increase inintracellular calcium concentration, increased expression of cellularadhesion molecules, and the promotion of leukocyte migration.Demonstration of the importance of the MCP-1/CCR-2 interaction has beenprovided by experiments with genetically modified mice. MCP-1−/− micewere unable to recruit monocytes into sites of inflammation afterseveral different types of immune challenge (Lu, B. et al., J. Exp.Med., 187:601 (1998)). Likewise, CCR-2−/− mice were unable to recruitmonocytes or produce interferon-γ when challenged with various exogenousagents; moreover, the leukocytes of CCR-2 null mice did not migrate inresponse to MCP-1 (Boring, L. et al., J. Clin. Invest., 100:2552(1997)), thereby demonstrating the specificity of the MCP-1/CCR-2interaction. Two other groups have independently reported equivalentresults with different strains of CCR-2−/− mice (Kuziel, W. A. et al.,Proc. Natl. Acad. Sci. USA, 94:12053 (1997), and Kurihara, T. et al., J.Exp. Med., 186:1757 (1997)). The viability and generally normal healthof the MCP-1−/− and CCR-2−/− animals is noteworthy, in that disruptionof the MCP-1/CCR-2 interaction does not induce physiological crisis.Taken together, these data lead one to the conclusion that moleculesthat block the actions of MCP-1/CCR-2 would be useful in treating anumber of inflammatory and autoimmune disorders (reviewed in: Feria, M.et al., Exp. Opin. Ther. Patents, 16:49 (2006); and Dawson, J. et al.,Exp. Opin. Ther. Targets, 7:35 (2003)). This hypothesis has now beenvalidated in a number of different animal disease models, as describedbelow.

It is known that MCP-1 is upregulated in patients with rheumatoidarthritis (Koch, A. et al., J. Clin. Invest., 90:772-779 (1992)).Moreover, several preclinical studies have demonstrated the potentialtherapeutic value of antagonism of the MCP-1/CCR-2 interaction intreating rheumatoid arthritis. A DNA vaccine encoding MCP-1 was shownrecently to ameliorate chronic polyadjuvant-induced arthritis in rats(Youssef, S. et al., J. Clin. Invest., 106:361 (2000)). Likewise, thedisease symptoms could be controlled via direct administration ofantibodies for MCP-1 to rats with collagen-induced arthritis (Ogata, H.et al., J. Pathol., 182:106 (1997)), or streptococcal cell wall-inducedarthritis (Schimmer, R. C. et al., J. Immunol., 160:1466 (1998)).Perhaps most significantly, a peptide antagonist of MCP-1, MCP-1(9-76),was shown both to prevent disease onset and to reduce disease symptoms(depending on the time of administration) in the MRL-lpr mouse model ofarthritis (Gong, J.-H. et al., J. Exp. Med., 186:131 (1997)). Moreover,it has been demonstrated the administration of small molecule CCR-2antagonists reduced clinical score in rodent models of arthritis(Brodmerkel, C. M. et al., J. Immunol., 175:5370 (2005); and Xia, M. etal., U.S. Publication No. 2006/0069123). Administration of an anti-CCR-2antibody had varying effects on murine CIA, depending on the time ofadministration (Bruhl, H. et al., J. Immunol., 172:890 (2004)). Recentstudies with CCR-2−/− mice have suggested that deletion of CCR-2 canexacerbate rodent arthritis models in specific experimentalcircumstances (Quinones, M. P. et al., J. Clin. Invest., 113:856 (2004);Quinones, M. P. et al., J. Mol. Med., 84:503 (2006)).

It is known that MCP-1 is upregulated in atherosclerotic lesions, and ithas been shown that circulating levels of MCP-1 are reduced throughtreatment with therapeutic agents (Rezaie-Majd, A. et al., Arterioscler.Thromb. Vasc. Biol., 22:1194-1199 (2002)). Several key studies havedemonstrated the potential therapeutic value of antagonism of theMCP-1/CCR-2 interaction in treating atherosclerosis. For example, whenMCP-1−/− mice are crossed with LDL receptor-deficient mice, an 83%reduction in aortic lipid deposition was observed (Gu, L. et al., Mol.Cell, 2:275 (1998)). Similarly, when MCP-1 was genetically ablated frommice which already overexpressed human apolipoprotein B, the resultingmice were protected from atherosclerotic lesion formation relative tothe MCP-1+/+ apoB control mice (Gosling, J. et al., J. Clin. Invest.,103:773 (1999)). Likewise, when CCR-2−/− mice are crossed withapolipoprotein E−/− mice, a significant decrease in the incidence ofatherosclerotic lesions was observed (Boring, L. et al., Nature, 394:894(1998); Dawson, T. C. et al., Atherosclerosis, 143:205 (1999)). Finally,when apolipoprotein E−/− mice are administered a gene encoding a peptideantagonist of CCR-2, then lesion size is decreased and plaque stabilityis increased (Ni, W. et al., Circulation, 103:2096-2101 (2001)).Transplantation of bone marrow from CCR-2−/− mice into ApoE3-Leiden miceinhibited early atherogenesis (Guo, J. et al., Arterioscler. Thromb.Vasc. Biol., 23:447 (2003)), but had minimal effects on advanced lesions(Guo, J. et al., Arterioscler. Thromb. Vasc. Biol., 25:1014 (2005)).

Patients with type 2 diabetes mellitus typically exhibit insulinresistance as one of the hallmark features of the disease. Insulinresistance is also associated with the grouping of abnormalities knownas the “metabolic syndrome” or “syndrome X,” which includes obesity,atherosclerosis, hypertension, and dyslipidemia (reviewed in: Eckel etal., Lancet, 365:1415 (2005)). It is well-recognized that inflammationplays a role in exacerbating the disease process in type 2 diabetes andthe “syndrome X” pathologies (reviewed in: Chen, H., PharmacologicalResearch, 53:469 (2006); Neels et al., J. Clin. Invest., 116:33 (2006);Danadona et al., Am. J. Cardiol., 90:27 G-33G (2002); Pickup et al.,Diabetologia, 41:1241 (1998)). MCP-1 is recognized as playing a role inobesity-induced insulin resistance. In culture, human preadipocytesconstitutively expressed MCP-1 (Gerhardt, Mol. Cell. Endocrinology,175:81 (2001)). CCR-2 is expressed on adipocytes; Addition of MCP-1 todifferentiated adipocytes in vitro decreases insulin-stimulated glucoseuptake and the expression of several adipogenic genes (LpL, adipsin,GLU-4), aP2, β3-adrenergic receptor, and PPARγ) (Sartipy, P. et al.,Proc. Natl. Acad. Sci. USA, 96:6902 (1999)). Patients with type 2diabetes had greater levels of circulating MCP-1 than non-diabeticcontrols (Nomura, S. et al., Clin. Exp. Immunol., 121:437 (2000)), andrelease of MCP-1 from adipose tissue could be reduced by treatment withanti-diabetic therapies such as metformin or thiazolidinediones (Bruun,J. M. et al., J. Clin. Endocrinol. Metab., 90:2282 (2005)). Likewise,MCP-1 was also overexpressed in murine experimental models of obesity,and was primarily produced by adipose tissue (Sartipy et al., Proc.Natl. Acad. Sci. USA, 100:7265 (2003)). In obese mice, the expression ofMCP-1 both preceded and occurred concurrently with the onset of insulinresistance (Xu, H. et al., J. Clin. Invest., 112:1821 (2003)). Anotherstudy showed that the expression of MCP-1 positively correlated withbody mass in the perigonadal adipose tissue of mice (Weisberg et al., J.Clin. Invest., 112:1796 (2003)). Consistent with these data, thedevelopment of insulin resistance in db/db mice was ameliorated eithervia genetic deletion of MCP-1 or by gene-induced expression of adominant negative peptide (Kanda, H. et al., J. Clin. Invest., 116:1494(2006)). The logical converse could also be demonstrated: overexpressionof MCP-1 in adipose tissue promoted insulin resistance (Kamei, N. etal., J. Biol. Chem., 281:26602 (2006)). One conflicting result showingthat genetic deletion of MCP-1 does not effect insulin resistance in thedb/db mouse has also appeared (Chow, F. Y. et al., Diabetologia, 50:471(2007)). Consistent with the data on MCP-1, direct studies with CCR-2(the MCP-1 receptor) have showed that it plays a role in the formationof obesity and obesity-induced insulin resistance. Maintenance of a highfat diet increased the numbers of circulating CCR-2⁺ inflammatorymonocytes in both wild-type (Tsou, C. L. et al., J. Clin. Invest.,117:902 (2007)) and ApoE−/− mice (Tacke, F. et al., J. Clin. Invest.,117:185 (2007)). Genetic deletion of CCR-2 reduced numbers of activatedmacrophages in murine adipose tissue (Lumeng, C. N. et al., Diabetes,56:16 (2007)), but did not affect a population of M2 adipose macrophagesthought to maintain the “lean” state (Lumeng, C. N. et al., J. Clin.Invest., 117:175 (2007)). Genetic deletion of CCR-2 reduced diet-inducedobesity and improved insulin sensitivity in diet-induced obesity model(Weisberg, S. P. et al., J. Clin. Invest., 116:115 (2006); Cornelius, P.et al., PCT Publication No. WO 2006/013427 A2), depending onexperimental conditions (Chen, A. et al., Obes. Res., 13:1311 (2005)).Administration of a small molecule CCR-2 antagonist also improvedinsulin sensitivity in this same model (Weisberg, S. P. et al., J. Clin.Invest., 116:115 (2006)).

Two studies described the important role of CCR-2 inhypertension-induced vascular inflammation, remodeling, and hypertrophy(Bush, E. et al., Hypertension, 36:360 (2000); Ishibashi, M. et al.,Circ. Res., 94:1203 (2004)).

It is known that MCP-1 is upregulated in human multiple sclerosis, andit has been shown that effective therapy with interferon β-1b reducesMCP-1 expression in peripheral blood mononuclear cells, suggesting thatMCP-1 plays a role in disease progression (Iarlori, C. et al., J.Neuroimmunol., 123:170-179 (2002)). Other studies have demonstrated thepotential therapeutic value of antagonism of the MCP-1/CCR-2 interactionin treating multiple sclerosis; all of these studies have beendemonstrated in experimental autoimmune encephalomyelitis (EAE), theconventional animal model for multiple sclerosis. Administration ofantibodies for MCP-1 to animals with EAE significantly diminisheddisease relapse (Kennedy, K. J. et al., J. Neuroimmunol., 92:98 (1998)).Furthermore, two reports have shown that CCR-2−/− mice are resistant toEAE (Fife, B. T. et al., J. Exp. Med., 192:899 (2000); Izikson, L. etal., J. Exp. Med., 192:1075 (2000)). A subsequent report extended theseinitial observations by examining the effects of CCR-2 deletion in micefrom different strains (Gaupp, S. et al., Am. J. Pathol., 162:139(2003)). Notably, administration of a small molecule CCR-2 antagonistalso blunted disease progression in C57BL/6 mice (Brodmerkel, C. M. etal., J. Immunol., 175:5370 (2005)).

It is known that MCP-1 is upregulated in patients who developbronchiolitis obliterans syndrome after lung transplantation(Reynaud-Gaubert, M. et al., J. Heart Lung Transplant., 21:721-730(2002); Belperio, J. et al., J. Clin. Invest., 108:547-556 (2001)). In amurine model of bronchiolitis obliterans syndrome, administration of anantibody to MCP-1 led to attenuation of airway obliteration; likewise,CCR-2−/− mice were resistant to airway obliteration in this same model(Belperio, J. et al., J. Clin. Invest., 108:547-556 (2001)). These datasuggest that antagonism of MCP-1/CCR-2 may be beneficial in treatingrejection of organs following transplantation. In addition, studies haveshown that disruption of MCP-1/CCR-2 axis was able to prolong thesurvival of islet transplant (Lee, I. et al., J. Immunol., 171:6929(2003); Abdi, R. et al., J. Immunol., 172:767 (2004)). In rat graftmodels, CCR-2 and MCP-1 was shown to be upregulated in grafts thatdevelop graft vasculopathy (Horiguchi, K. et al., J. Heart LungTransplant., 21:1090 (2002)). In another study, anti-MCP-1 gene therapyattenuated graft vasculopathy (Saiura, A. et al., Arterioscler. Thromb.Vasc. Biol., 24:1886 (2004)). One study described inhibition ofexperimental vein graft neoinitimal formation by blockage of MCP-1(Tatewaki, H. et al., J. Vasc. Surg., 45:1236 (2007)).

Other studies have demonstrated the potential therapeutic value ofantagonism of the MCP-1/CCR-2 interaction in treating asthma.Sequestration of MCP-1 with a neutralizing antibody inovalbumin-challenged mice resulted in marked decrease in bronchialhyperresponsiveness and inflammation (Gonzalo, J.-A. et al., J. Exp.Med., 188:157 (1998)). It proved possible to reduce allergic airwayinflammation in Schistosoma mansoni egg-challenged mice through theadministration of antibodies for MCP-1 (Lukacs, N. W. et al., J.Immunol., 158:4398 (1997)). Consistent with this, MCP-1−/− micedisplayed a reduced response to challenge with Schistosoma mansoni egg(Lu, B. et al., J. Exp. Med., 187:601 (1998)).

Other studies have demonstrated the potential therapeutic value ofantagonism of the MCP-1/CCR-2 interaction in treating kidney disease.Administration of antibodies for MCP-1 in a murine model ofglomerularnephritis resulted in a marked decrease in glomerular crescentformation and deposition of type I collagen (Lloyd, C. M. et al., J.Exp. Med., 185:1371 (1997)). In addition, MCP-1−/− mice with inducednephrotoxic serum nephritis showed significantly less tubular damagethan their MCP-1+/+ counterparts (Tesch, G. H. et al., J. Clin. Invest.,103:73 (1999)).

Several studies have demonstrated the potential therapeutic value ofantagonism of the MCP-1/CCR-2 interaction in treating systemic lupuserythematosus. CCR-2−/− mice exhibited prolonged survival and reducedrenal disease relative to their WT counterparts in a murine model ofsystemic lupus erythematosus (Perez de Lema, G. et al., J. Am. Soc.Neph., 16:3592 (2005)). These data are consistent with thedisease-modifying activity found in recent studies on genetic deletionof MCP-1 (Shimizu, S. et al., Rheumatology (Oxford), 43:1121 (2004);Tesch, G. H. et al., J. Exp. Med., 190:1813 (1999)) or administration ofa peptide antagonist of CCR-2 (Hasegawa, H. et al., Arthritis Rheum.,48:2555 (2003)) in rodent models of lupus.

A remarkable 30-fold increase in CCR-2⁺ lamina propria lymphocytes wasobserved in the small bowels from Crohn's patients relative tonon-diseased ileum (Connor, S. J. et al., Gut, 53:1287 (2004)). Also ofnote, there was an expansion in the subset of circulatingCCR-2⁺/CD14⁺/CD56⁺ monocytes in patients with active Crohn's diseaserelative to controls. Several rodent studies have demonstrated thepotential therapeutic value of antagonism of the MCP-1/CCR-2 interactionin treating Crohn's disease/colitis. CCR-2−/− mice were protected fromthe effects of dextran sodium sulfate-induced colitis (Andres, P. G. etal., J. Immunol., 164:6303 (2000)). Administration of a small moleculeantagonist of CCR-2, CCR-5, and CXCR3 (murine binding affinities=24,236, and 369 nM, respectively) also protected against dextran sodiumsulfate-induced colitis (Tokuyama, H. et al., Int. Immunol., 17:1023(2005)). Finally, MCP-1−/− mice showed substantially reduced colonicdamage (both macroscopic and histological) in a hapten-induced model ofcolitis (Khan, W. I. et al., Am. J. Physiol. Gastrointest. LiverPhysiol., 291:G803 (2006)).

Two reports described the overexpression of MCP-1 in the intestinalepithelial cells and bowel mucosa of patients with inflammatory boweldisease (Reinecker, H. C. et al., Gastroenterology, 108:40 (1995), andGrimm, M. C. et al., J. Leukoc. Biol., 59:804 (1996)).

One study described the association of promoter polymorphism in theMCP-1 gene with scleroderma (systemic sclerosis) (Karrer, S. et al., J.Invest. Dermatol., 124:92 (2005)). In related models of tissue fibrosis,inhibition of CCR-2/MCP-1 axis reduced fibrosis in skin (Yamamoto, T. etal., J. Invest. Dermatol., 121:510 (2003); Ferreira, A. M. et al., J.Invest. Dermatol., 126:1900 (2006)), lung (Okuma, T. et al., J. Pathol.,204:594 (2004); Gharaee-Kermani, M. et al., Cytokine, 24:266 (2003)),kidney (Kitagawa, K. et al., Am. J. Pathol., 165:237 (2004); Wada, T. etal., J. Am. Soc. Nephrol., 15:940 (2004)), heart (Hayashidani, S. etal., Circulation, 108:2134 (2003)), and liver (Tsuruta, S. et al., Int.J. Mol. Med., 14:837 (2004)).

One study has demonstrated the potential therapeutic value of antagonismof the MCP-1/CCR-2 interaction in treating alveolitis. When rats withIgA immune complex lung injury were treated intravenously withantibodies raised against rat MCP-1 (JE), the symptoms of alveolitiswere partially alleviated (Jones, M. L. et al., J. Immunol., 149:2147(1992)).

Several studies have shown the potential therapeutic value of antagonismof the MCP-1/CCR-2 interaction in treating cancer (reviewed in: Craig,M. J. et al., Cancer Metastasis Rev., 25:611 (2006); Conti, I. et al.,Seminars in Cancer Biology, 14:149 (2004); Giles, R. et al., Curr.Cancer Drug Targets, 6:659 (2006)). When immunodeficient mice bearinghuman breast carcinoma cells were treated with an anti-MCP-1 antibody,inhibition of lung micrometastases and increases in survival wereobserved (Salcedo, R. et al., Blood, 96:34-40 (2000)). Using humanclinical tumor specimens, CCR-2 expression was associated with prostratecancer progression (Lu, Y. et al., J. Cell. Biochem., 101:676 (2007)).In vitro, MCP-1 expression has been shown to mediate prostrate cancercell growth and invasion (Lu, Y. et al., Prostate, 66:1311 (2006));furthermore, MCP-1 expressed by prostate cancer cells induced human bonemarrow progenitors for bone resorption (Lu, Y. et al., Cancer Res.,67:3646 (2007)).

Multiple studies have described the potential therapeutic value ofantagonism of the MCP-1/CCR-2 interaction in treating restenosis. Inhumans, MCP-1 levels correlate directly with risk for restenosis(Cipollone, F. et al., Arterioscler. Thromb. Vasc. Biol., 21:327(2001)). Mice deficient in CCR-2 or in MCP-1 showed reductions in theintimal area and in the intima/media ratio (relative to wildtypelittermates) after arterial injury (Roque, M. et al., Arterioscler.Thromb. Vasc. Biol., 22:554 (2002); Schober, A. et al., Circ. Res.,95:1125 (2004); Kim, W. J. et al., Biochem. Biophys. Res. Commun.,310:936 (2003)). In mice, transfection of a dominant negative inhibitorof MCP-1 in the skeletal muscle (Egashira, K. et al., Circ. Res.,90:1167 (2002)) also reduced intimal hyperplasia after arterial injury.Blockade of CCR-2 using a neutralizing antibody reduced neointimalhyperplasia after stenting in primates (Horvath, C. et al., Circ. Res.,90:488 (2002)).

Two reports describe the overexpression of MCP-1 rats with induced braintrauma (King, J. S. et al., J. Neuroimmunol., 56:127 (1994), and Berman,J. W. et al., J. Immunol., 156:3017 (1996)). In addition, studies haveshown that both CCR-2−/− (Dimitrijevic, O. B. et al., Stroke, 38:1345(2007)) and MCP-1−/− mice (Hughes, P. M. et al., J. Cereb. Blood FlowMetab., 22:308 (2002)) are partially protected from ischemia/reperfusioninjury.

It is known that monocytes/macrophages play an important role in thedevelopment of neuropathic pain (Liu, T. et al., Pain, 86:25 (2000)).Consistent with this notion, a potential role for CCR-2 in the treatmentof both inflammatory and neuropathic pain has been described recently.CCR-2−/− mice showed altered responses to inflammatory pain relative totheir WT counterparts, including reduced pain behavior afterintraplantar formalin injection and slightly reduced mechanicalallodynia after intraplantar CFA injection (Abbadie, C. et al., Proc.Natl. Acad. Sci. USA, 100:7947 (2003)). In addition, CCR-2−/− mice didnot display significant mechanical allodynia after sciatic nerve injury.Likewise, a small molecule CCR-2 antagonist reduced mechanical allodyniato ˜80% of pre-injury levels after oral administration (Abbadie, C. etal., PCT Publication No. WO 2004/110376).

One study described the critical role of MCP-1 in ischemiccardiomyopathy (Frangogiannis, N. G. et al., Circulation, 115:584(2007)). Another study described the attenuation of experimental heartfailure following inhibition of MCP-1 (Hayashidani, S. et al.,Circulation, 108:2134 (2003)).

Other studies have provided evidence that MCP-1 is overexpressed invarious disease states not mentioned above. These reports providecorrelative evidence that MCP-1 antagonists could be useful therapeuticsfor such diseases. Another study has demonstrated the overexpression ofMCP-1 in rodent cardiac allografts, suggesting a role for MCP-1 in thepathogenesis of transplant arteriosclerosis (Russell, M. E. et al.,Proc. Natl. Acad. Sci. USA, 90:6086 (1993)). The overexpression of MCP-1has been noted in the lung endothelial cells of patients with idiopathicpulmonary fibrosis (Antoniades, H. N. et al., Proc. Natl. Acad. Sci.USA, 89:5371 (1992)). Similarly, the overexpression of MCP-1 has beennoted in the skin from patients with psoriasis (Deleuran, M. et al., J.Dermatol. Sci., 13:228 (1996), and Gillitzer, R. et al., J. Invest.Dermatol., 101:127 (1993)); correlative findings with predominance ofCCR-2+ cells have also been reported (Vestergaard, C. et al., Acta Derm.Venerol., 84:353 (2004)). Finally, a recent report has shown that MCP-1is overexpressed in the brains and cerebrospinal fluid of patients withHIV-1-associated dementia (Garzino-Demo, A., PCT Publication No. WO99/46991).

In addition, CCR-2 polymorphism has been shown to be associated withsarcoidosis at least in one subset of patients (Spagnolo, P. et al., Am.J. Respir. Crit. Care Med., 168:1162 (2003)).

It should also be noted that CCR-2 has been implicated as a co-receptorfor some strains of HIV (Doranz, B. J. et al., Cell, 85:1149 (1996)). Ithas also been determined that the use of CCR-2 as an HIV co-receptor canbe correlated with disease progression (Connor, R. I. et al., J. Exp.Med., 185:621 (1997)). This finding is consistent with the recentfinding that the presence of a CCR-2 mutant, CCR-2-64I, is positivelycorrelated with delayed onset of HIV in the human population (Smith, M.W. et al., Science, 277:959 (1997)). Although MCP-1 has not beenimplicated in these processes, it may be that MCP-1 antagonists that actvia binding to CCR-2 may have beneficial therapeutic effects in delayingthe disease progression to AIDS in HIV-infected patients.

It should be noted that CCR-2 is also the receptor for the humanchemokines MCP-2, MCP-3, and MCP-4 (Luster, New Eng. J. Med.,338:436-445 (1998)). Since the new compounds of formula (I) describedherein antagonize MCP-1 by binding to the CCR-2 receptor, it may be thatthese compounds of formula (I) are also effective antagonists of theactions of MCP-2, MCP-3, and MCP-4 that are mediated by CCR-2.Accordingly, when reference is made herein to “antagonism of MCP-1,” itis to be assumed that this is equivalent to “antagonism of chemokinestimulation of CCR-2.”

Accordingly, compounds that modulate chemokine activity coulddemonstrate a wide range of utilities in treating inflammatory,allergic, autoimmune, metabolic, cancer and/or cardiovascular diseases.PCT Publication Nos. WO 2005/021500 A1 (incorporated herein by referenceand assigned to present applicant), WO 2008/014381 A1, WO 2008/014360 A1and WO 2008/014361 A1, disclose compounds that modulate MCP-1, MCP-2,MCP-3 and MCP-4 activity via CCR-2. The references also disclose variousprocesses to prepare these compounds including multistep syntheses thatinclude the introduction and subsequent removal of protecting groups.

It is desirable to find new compounds with improved pharmacologicalcharacteristics compared with known chemokine modulators. For example,it is desirable to find new compounds with equipotent dual CCR-2/5inhibitory activity in comparison to selectivity for CCR-2 alone,predominantly CCR-2 versus CCR-5, predominantly CCR-5 versus CCR-2, orother G protein-coupled receptors (i.e., 5HT2A receptor). It is alsodesirable to find compounds with equipotent dual CCR-2/5 inhibitoryactivity and advantageous characteristics in one or more of thefollowing categories:

(a) pharmaceutical properties (i.e., solubility, permeability,amenability to sustained release formulations);

(b) dosage requirements (e.g., lower dosages and/or once-daily dosing);

(c) factors which decrease blood concentration peak-to-troughcharacteristics (i.e., clearance and/or volume of distribution);

(d) factors that increase the concentration of active drug at thereceptor (i.e., protein binding, volume of distribution);

(e) factors that decrease the liability for clinical drug-druginteractions (cytochrome P450 enzyme inhibition or induction, such asCYP 2D6 inhibition, see Dresser, G. K. et al., Clin. Pharmacokinet.,38:41-57 (2000), which is hereby incorporated by reference); and

(f) factors that decrease the potential for adverse side-effects (e.g.,pharmacological selectivity beyond G protein-coupled receptors,potential chemical or metabolic reactivity, limited CNS penetration,ion-channel selectivity). It is especially desirable to find compoundshaving a desirable combination of the aforementioned pharmacologicalcharacteristics.

It is also desirable in the art to provide new and/or improved processesto prepare such compounds. These processes may be characterized, withoutlimitation, by a) facile adaptation to larger scale production, such aspilot plant or manufacturing scales; b) process steps and/or techniquesenabling improvements in the purity (including chiral purity), stabilityand/or ease of handling of intermediates and/or final compounds; and/orc) fewer process steps.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a novel antagonist:N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,or a pharmaceutically acceptable salt, solvate or prodrug, thereof,having unexpected equipotent dual CCR-2 and CCR-5 receptor inhibitoryactivity. In addition, the present invention presents a novel andunexpected combination of equipotent dual CCR-2/5 activity and desirablepharmacological characteristics. Crystalline and metabolite forms of thepresent invention are also provided. Pharmaceutical compositionscontaining the same and methods of using the same as agents for thetreatment of inflammatory diseases, allergic, autoimmune, metabolic,cancer and/or cardiovascular diseases is also an objective of thisinvention. The present disclosure also provides a process for preparingcompounds of Formula (I), includingN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide:

wherein R¹, R⁸, R⁹, R¹⁰, and

are as described herein. Compounds that are useful intermediates of theprocess are also provided herein.

The present disclosure also provides the use ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-(2-hydroxy-1,1-dimethyl-ethyl)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,or a pharmaceutically acceptable salt, solvate or prodrug, for themanufacture of a medicament for the treatment of inflammatory diseases,allergic, autoimmune, metabolic, cancer and/or cardiovascular diseases.

The present disclosure also provides metabolites of active compounds, orpharmaceutically acceptable salts or prodrugs thereof, pharmaceuticalcompositions thereof and methods of using the metabolites in thetreatment of inflammatory, allergic, autoimmune, metabolic, cancerand/or cardiovascular diseases, particularly diabetes, multiplesclerosis, Crohn's disease and/or atherosclerosis.

Accordingly, disclosed herein are novel modulators of chemokineactivity, or pharmaceutically acceptable salts or prodrugs thereof,having an unexpected combination of desirable pharmacologicalcharacteristics.

The present disclosure provides pharmaceutical compositions comprising apharmaceutically acceptable carrier and a therapeutically effectiveamount of at least one of the compounds of the present invention or apharmaceutically acceptable salt or prodrug form thereof.

The present disclosure also provides methods for treating inflammatory,allergic, autoimmune, metabolic, cancer and/or cardiovascular diseases,particularly diabetes, multiple sclerosis, Crohn's disease and/oratherosclerosis, comprising administering to a host in need of suchtreatment a therapeutically effective amount of at least one of thecompounds of the present invention, or a pharmaceutically acceptablesalt or prodrug form thereof.

The present disclosure provides a process for preparing compoundsdisclosed herein and intermediates useful therefore.

The present disclosure provides for the use of the compounds of thepresent invention in therapy.

The present disclosure provides the use of compounds of the presentinvention for the manufacture of a medicament for the treatment ofinflammatory, allergic, autoimmune, metabolic, cancer and/orcardiovascular diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 discloses the experimental and simulated powder patterns of theN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide.

FIG. 2 discloses the differential scanning calorimetry analysis ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,free base Form N-1.

FIG. 3 discloses the thermogravimetric analysis ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,free base Form N-1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel antagonist:N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,or a pharmaceutically acceptable salt, solvate or prodrug, thereof,having unexpected equipotent dual CCR-2 and CCR-5 receptor activity.Additionally, the present invention provides a novel combination ofdesirable pharmacological characteristics. Crystalline forms andmetabolites of the present invention are also provided. Pharmaceuticalcompositions containing the same and methods of using the same as agentsfor the treatment of inflammatory diseases, allergic, autoimmune,metabolic, cancer and/or cardiovascular diseases is also an objective ofthis invention. The present disclosure also provides a process forpreparing compounds of Formula (I), includingN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide:

wherein R¹, R⁸, R⁹, R¹⁰, and

are as described herein. Compounds that are useful intermediates of theprocess are also provided herein.

N-((1R,2S,5R)-5-(tert-Butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,unexpectedly demonstrates equipotent dual CCR-2/5 receptor inhibitoryactivity.

Additionally,N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamidedemonstrates a desirable combination of equipotent dual CCR-2/5 receptorinhibitory activity and pharmacological characteristics including asurprisingly high degree of oral bioavailability in combination withindications that it is highly efficacious and has excellent safetycriteria.

Known modulators of chemokine receptors, such as those disclosed in PCTPublication Nos. WO 2004/071460 A1 and WO 2005/021500 A1 (U.S. Pat. No.7,163,937, issued Jan. 16, 2007, assigned to present Applicant) are notsufficiently efficacious, as measured by their CCR-2 or CCR-5-bindingability (a measure of efficacy), and/or they lack appropriate criteriafor safety as indicated by ion channel selectivity as measured by hERGand Na+ ion channel studies.

Other known modulators of chemokine receptors, such as those disclosedin PCT Publication Nos. WO 2008/014381 A1, WO 2008/014360 A1 and WO2008/014361 A1, are selective as antagonist or partialagonist/antagonist of CCR-2 receptor activity. However, these knownmodulators demonstrate predominantly CCR-2 activity and are notequipotent dual antagonists, as measured by their CCR-2 andCCR-5-binding ability.

Other known modulators of chemokine receptors, such as those disclosedby Carter et al. (American Chemical Society, Aug. 17, 2008) are dualCCR-2/5 modulators but lack appropriate criteria for safety as indicatedby ion channel selectivity as measured by hERG and Na+ ion channelstudies.

In contrast, as illustrated by the data presented herein in the sectiontitled “Comparative Pharmacological Characteristics”, infra,N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamidesurprisingly exhibits equipotent CCR-5 and CCR-2 binding ability. Inaddition,N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamidedemonstrates a surprisingly high degree of membrane permeability, andyet maintains equipotent CCR-2/5 dual binding ability along withexcellent ion channel selectivity.

Accordingly, the present invention provides new chemokine modulatorshaving equipotent CCR-2 and CCR-5 binding ability and improvespharmacological characteristics that are expected to be useful intreating inflammatory, allergic, autoimmune, metabolic, cancer and/orcardiovascular diseases.

Embodiments

In one embodiment, the disclosure is directed toN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide:

and pharmaceutically acceptable salts, thereof.

Another embodiment is a crystalline form ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide.

Another embodiment is a crystalline form ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,wherein the crystalline form comprises the N-1 and/or P-1 Form.

Another embodiment is a crystalline form ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,wherein the crystalline form comprises the N-1 Form.

Another embodiment is a crystalline form ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,wherein the crystalline form comprises the N-1 or P-1 Form insubstantially pure form.

Another embodiment is a crystalline form ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,wherein the crystalline form comprises the N-1 Form in substantiallypure form.

Another embodiment is the N-1 Form characterized by unit cell parameterssubstantially equal to the following:

Cell dimensions:

-   -   a=7.3085(6)    -   b=16.257(1)    -   c=22.688(2)°    -   α°=90    -   β°=90    -   γ°=90

Space group P2₁2₁2₁

Molecules/unit cell (Z): 1

Density, calc g-cm⁻³: 1.194

wherein said crystal is at a temperature of about −70° C.

Another embodiment is the N-2 Form characterized by (or having) a powerx ray diffraction pattern substantially according to FIG. 1.

Another embodiment is the N-2 Form characterized by (or having) adifferential scanning calorimetry thermogram substantially in accordancewith that shown in FIG. 2, having an endothermic transition above ca.205° C.

Another embodiment is characterized by (or having) a thermal gravimetricanalysis curve in accordance with that shown in FIG. 3.

Another embodiment is a crystalline form ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,comprising Form N-1, characterized by the unit cell parameters found inTable 1; and/or a powder x-ray diffraction pattern substantiallyaccording to FIG. 1.

Another embodiment is a pharmaceutical composition comprised ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a pharmaceutical composition comprised ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,pharmaceutically acceptable salts, thereof, and a pharmaceuticallyacceptable carrier.

Another embodiment is a pharmaceutical composition comprised ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,pharmaceutically acceptable salts, thereof, and at least one additionaltherapeutic agent.

Another embodiment is a method for modulating chemokine or chemokinereceptor activity comprising administering to a patient in need thereofa therapeutically effective amount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of modulating CCR-2 and CCR-5 receptoractivity comprising administering to a patient in need thereof atherapeutically effective amount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method modulating MCP-1, MCP-2, MCP-3, MCP-4,MCP-5, MIP-1a, MIP-1b and RANTES activity that is mediated by the CCR-2and CCR-5 receptor comprising administering to a patient in need thereofa therapeutically effective amount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of modulating MCP-1 activity comprisingadministering to a patient in need thereof a therapeutically effectiveamount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating a disorder, comprisingadministering to a patient in need thereof a therapeutically effectiveamount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof, said the disorder isselected from diabetes, obesity, metabolic syndrome, stroke, neuropathicpain, ischemic cardiomyopathy, psoriasis, hypertension, scleroderma,osteoarthritis, aneurism, fever, cardiovascular disease, Crohn'sdisease, congestive heart failure, autoimmune diseases, HIV-infection,HIV-associated dementia, psoriasis, idiopathic pulmonary fibrosis,transplant arteriosclerosis, physically- or chemically-induced braintrauma, inflammatory bowel disease, alveolitis, colitis, systemic lupuserythematosus, nephrotoxic serum nephritis, glomerulonephritis, asthma,multiple sclerosis, atherosclerosis, vasculitis, vulnerable plaques,rheumatoid arthritis, restenosis, venous neointimal hyperplasia,dialysis-graft neointimal hyperplasia, arterio-venous shunt intimalhyperplasia, organ transplantation, chronic allograft nephropathy, andcancer.

Another embodiment is a method of treating a disorder, comprisingadministering to a patient in need thereof a therapeutically effectiveamount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof, wherein said disorder isselected from diabetes, obesity, Crohn's disease, psoriasis, idiopathicpulmonary fibrosis, transplant arteriosclerosis, physically- orchemically-induced brain trauma, inflammatory bowel disease, alveolitis,colitis, systemic lupus erythematosus, nephrotoxic serum nephritis,glomerulonephritis, asthma, multiple sclerosis, atherosclerosis, andrheumatoid arthritis, restenosis, organ transplantation, cancer, venousneointimal hyperplasia, dialysis-graft neointimal hyperplasia, andarterio-venous shunt neointimal hyperplasia.

Another embodiment is a method of treating a disorder, comprisingadministering to a patient in need thereof a therapeutically effectiveamount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof, wherein said disorder isselected from diabetes, obesity, Crohn's disease, systemic lupuserythematosus, glomerulonephritis, multiple sclerosis, atherosclerosis,restenosis, organ transplantation, venous neointimal hyperplasia,dialysis-graft neointimal hyperplasia, and arterio-venous shuntneointimal hyperplasia.

Another embodiment is a method of treating a disorder, comprisingadministering to a patient in need thereof a therapeutically effectiveamount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof, wherein said disorder isselected from multiple sclerosis, atherosclerosis, Crohn's disease,diabetes, venous neointimal hyperplasia, dialysis-graft neointimalhyperplasia, and arterio-venous shunt neointimal hyperplasia.

Another embodiment is a method of treating a disorder, comprisingadministering to a patient in need thereof a therapeutically effectiveamount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof, wherein said disorder isbeing selected from restenosis, organ transplantation, cancer, venousneointimal hyperplasia, dialysis-graft neointimal hyperplasia, andarterio-venous shunt neointimal hyperplasia.

Another embodiment is a method of treating diabetes, comprisingadministering to a patient in need thereof a therapeutically effectiveamount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating Crohn's disease, comprisingadministering to a patient in need thereof a therapeutically effectiveamount of tN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating multiple sclerosis,comprising administering to a patient in need thereof a therapeuticallyeffective amount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating atherosclerosis, comprisingadministering to a patient in need thereof a therapeutically effectiveamount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating restenosis, comprisingadministering to a patient in need thereof a therapeutically effectiveamount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating organ transplantation,comprising administering to a patient in need thereof a therapeuticallyeffective amount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating cancer, for example, breastcancer, liver cancer, prostate cancer and melanoma, comprisingadministering to a patient in need thereof a therapeutically effectiveamount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating venous neointimalhyperplasia, comprising administering to a patient in need thereof atherapeutically effective amount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating dialysis-graft neointimalhyperplasia, comprising administering to a patient in need thereof atherapeutically effective amount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating arterio-venous shuntneointimal hyperplasia, comprising administering to a patient in needthereof a therapeutically effective amount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating an inflammatory disease,allergic, autoimmune, metabolic, and/or cardiovascular diseasescomprising administering to a patient in need thereof a therapeuticallyeffective amount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of treating a disease which is at leastpartially mediated by CCR-2 and CCR-5, comprising administering to apatient in need thereof a therapeutically effective amount ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideand pharmaceutically acceptable salts, thereof.

Another embodiment is a method of usingN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,and pharmaceutically acceptable salts, thereof, in the preparation of amedicament for the treatment of diabetes, obesity, metabolic syndrome,stroke, neuropathic pain, ischemic cardiomyopathy, psoriasis,hypertension, scleroderma, osteoarthritis, aneurism, fever,cardiovascular disease, Crohn's disease, congestive heart failure,autoimmune diseases, HIV-infection, HIV-associated dementia, psoriasis,idiopathic pulmonary fibrosis, transplant arteriosclerosis, physically-or chemically-induced brain trauma, inflammatory bowel disease,alveolitis, colitis, systemic lupus erythematosus, nephrotoxic serumnephritis, glomerulonephritis, asthma, multiple sclerosis,atherosclerosis, vasculitis, vulnerable plaques, rheumatoid arthritis,restenosis, venous neointimal hyperplasia, dialysis-graft neointimalhyperplasia, arterio-venous shunt intimal hyperplasia, organtransplantation, chronic allograft nephropathy, cancer, venousneointimal hyperplasia, dialysis-graft neointimal hyperplasia, andarterio-venous shunt neointimal hyperplasia.

In yet another embodiment, the disclosure is directed toN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-(2-hydroxy-1,1-dimethyl-ethyl)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide:

and pharmaceutically acceptable salts, thereof. Pharmaceuticalcompositions containing the same and methods of using the same as agentsfor the treatment of inflammatory diseases, allergic, autoimmune,metabolic, cancer and/or cardiovascular diseases is also an embodimentof this invention.

Process Embodiments

In a first embodiment, the disclosure provides a process for preparing acompound of formula I, or a salt form thereof:

comprising:

1) converting a hydrazone compound of formula I-cc to a compound offormula I-bb; and

2) coupling the compound of formula I-bb with a compound of formulaI-aa; wherein:

-   -   R₁ is independently hydrogen or an amine-protecting group        selected from a carbobenzyloxy group, a tert-butyloxycarbonyl, a        fluorenylmethyloxycarbonyl group, a benzyl group, and a        p-methoxybenzyl group;    -   R₈ and R₉ are independently hydrogen or C₁₋₆alkyl;    -   R₂₁ is ═O;    -   HET is an optionally substituted 3- to 14-membered heterocyclo        or heteroaryl bicyclic ring having at least one nitrogen        heteroatom; and    -   LG is —OR₁₆, wherein R₁₆ is C₁₋₆alkyl, phenyl, a 5- to        7-membered heteroaryl having one or more atoms selected from N,        S, or O, or a 3- to 7-membered cycloalkyl, all of which are        optionally substituted with 1 to 3 groups selected from halogen,        CF₃ or C₁₋₆alkyl.

In a second embodiment, the disclosure provides a process wherein thecompound of formula I isN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide.

In a third embodiment, the disclosure provides a process for preparing acompound of formula I wherein the compound of formula I-bb is a compoundof the formula:

In a fourth embodiment, the disclosure provides a process for preparinga compound of formula I wherein the coupling occurs via an acidicwork-up followed by the addition of a base.

In a fifth embodiment, the disclosure provides a process for preparing acompound of formula I wherein the acid in acidic work-up is selectedfrom citric acid, tartaric acid, glycolic acid, and hydrochloric acid.

In a sixth embodiment, the disclosure provides a process for preparing acompound of formula I wherein the base is selected from K₂HPO₄, Na₂HPO₄,NaHCO₃ and KHCO₃.

In a seventh embodiment, the disclosure provides a process for preparinga compound of formula I wherein converting of the hydrazone compound offormula I-cc to the compound of formula I-bb comprises reacting thehydrazone compound of formula I-cc with an acylating agent, anucleophile, and/or a tertiary amine base in a solvent followed byinterception with either (i) an alcohol in the presence of a secondtertiary amine base or (ii) an alkoxide to form the compound of formulaI-bb.

In an eighth embodiment, the disclosure provides a process wherein theacylating agent is selected from phosphorus oxychloride, oxalylchloride, thionyl chloride and phosgene, preferably phosphorusoxychloride.

In a ninth embodiment, the disclosure provides a process wherein thetertiary amine base is selected from triethylamine,N—N-diisopropyl-N-ethyl amine, tri-n-propylamine, N-methyl morpholineand 1,4-diazabicyclo[2.2.2]octane, preferably N—N-diisopropyl-N-ethylamine.

In a tenth embodiment, the disclosure provides a process wherein thenucleophile is selected from dimethyl 4-aminopyridine, dimethylaniline,pyridine and lutidine, preferably dimethyl 4-aminopyridine.

In an eleventh embodiment, the disclosure provides a process wherein theamount of nucleophile used in the reaction is 2.0 to 4.0 equivalents,preferably, 3.0 equivalents.

In a twelfth embodiment, the disclosure provides a process wherein thealcohol or alkoxide is selected from phenol, pentafluorophenol,methanol, ethanol, sodium methoxide and sodium phenolate, preferably,phenol.

In a thirteenth embodiment, the present disclosure provides a processwherein the second tertiary amine base is selected from triethylamine,N—N-diisopropyl-N-ethyl amine, tri-n-propylamine, N-methyl morpholine,and 1,4-diazabicyclo[2.2.2]octane, preferably, N—N-diisopropyl-N-ethylamine.

In a fourteenth embodiment, the disclosure provides a process whereinthe solvent is selected from acetonitrile, dichloromethane and neatphosphorus oxychloride.

In a fifteenth embodiment, the disclosure provides a process wherein thereaction and interception is carried out at a temperature in the rangeof ambient temperature to 70° C.

In a sixteenth embodiment, the disclosure provides a process wherein thecompound of formula I-cc is a compound of the formula:

In a seventeenth embodiment, the present invention provides a processwherein the hydrazone compound formula I-cc is prepared by a processcomprising reacting a hydrazone compound of formula I-dd

with orthoformate in the presence of an acid at elevated temperature toyield the hydrazone compound of formula I-cc; wherein R₂₁ is ═O; R₂₂ is—NH—NH₂; and R₂₃ is cyanoalkyl; or R₂₂ and R₂₃ may be taken together toform a 5- to 8-membered ring, wherein the ring may be optionallysubstituted with one or more substituents selected from amino, alkyl,aryl, or heteroaryl and may optionally contain 1, 2, 3, or 4 heteroatomsindependently selected from the group consisting of N, S, or O.

In an eighteenth embodiment, the disclosure provides a process whereinthe orthoformate is selected from trimethylorthoformate,triethylorthoformate, and tripropylorthoformate, preferably,trimethylorthoformate.

In a nineteenth embodiment, the disclosure provides a process whereinthe acid is selected from acetic acid, trifluoroacetic acid,hydrochloric acid and methanesulfonic acid, preferably, acetic acid.

In a twentieth embodiment, the disclosure provides a process wherein thereaction is carried out at a temperature in the range of 40-90° C.,preferably, 40-75° C.

In a twenty-first embodiment, the disclosure provides a process whereinthe compound of formula I-dd is selected from a compound of the formula:

In a twenty-second embodiment, the disclosure provides a process whereinthe hydrazone compound formula I-dd is prepared by a process comprisingcondensing a compound of formula I-ee, R₂₃═O, with a carbazide compoundof formula I-ff:

in a solvent in the presence of a base to yield the hydrazone compoundof formula I-dd; wherein R₂₁ is ═O and R₂₂ and R₂₃ are as set forthabove.

In a twenty-third embodiment, the disclosure provides a process whereinthe solvent is selected from ethanol, 2-propanol, 1-propanol andmethanol, preferably, ethanol.

In a twenty-fourth embodiment, the disclosure provides a process whereinthe base is selected from sodium acetate, potassium acetate,triethylamine and N—N-diisopropyl-N-ethyl amine, preferably, sodiumacetate.

In a twenty-fifth embodiment, the disclosure provides a process whereinthe condensation occurs at a temperature in the range of 20-60° C.,preferably, 25-55° C.

In a twenty-sixth embodiment, the present invention provides a processwherein the carbazide compound of formula I-ff is semicarbazidehydrochloride.

In a twenty-seventh embodiment, the present invention provides a processwherein the compound of formula I-ee is pivaloyl acetonitrile.

In a twenty-eighth embodiment, the present invention provides a processfor preparingN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamidecomprising the process set forth in the following scheme:

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof. Thus, theabove embodiments should not be considered limiting. Any and allembodiments of the present invention may be taken in conjunction withany other embodiment or embodiments to describe additional embodiments.Each individual element (e.g., preferable or special aspects) of theembodiments is its own independent embodiment. Furthermore, any elementof an embodiment is meant to be combined with any and all other elementsfrom any embodiment to describe an additional embodiment. In addition,the present invention encompasses combinations of different embodiment,parts of embodiments, definitions, descriptions, and examples of theinvention noted herein.

Definitions

The following are definitions of terms used in this specification andappended claims. The initial definition provided for a group or termherein applies to that group or term throughout the specification andclaims, individually or as part of another group, unless otherwiseindicated.

The term “alkyl” refers to straight or branched chain hydrocarbon groupshaving 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms. Whennumbers appear in a subscript after the symbol “C”, the subscriptdefines with more specificity the number of carbon atoms that aparticular group may contain. For example, “C₁₋₆alkyl” refers tostraight and branched chain alkyl groups with 1 to 6 carbon atoms, suchas methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, andso forth. The subscript “0” refers to a bond. Thus, the termhydroxy(C₀₋₂)alkyl or (C₀₋₂)hydroxyalkyl includes hydroxy, hydroxymethyland hydroxyethyl. Alkyl groups may be substituted with 1 to 3 groupsselected from (C₁₋₆)alkyl, (C₂₋₆)alkenyl, hydroxy, halogen, cyano,nitro, CF₃, O(C₁₋₆alkyl), OCF₃, C(═O)H, C(═O)(C₁₋₆alkyl), CO₂H,CO₂(C₁₋₆alkyl), NHCO₂(C₁₋₆alkyl), —S(C₁₋₆alkyl), NH₂, NH(C₁₋₆alkyl),N(C₁₋₆alkyl)₂, N(CH₃)₃ ⁺, SO₂(C₁₋₆alkyl), C(═O)(C₁₋₄alkylene)NH₂,C(═O)(C₁₋₄alkylene)NH(alkyl), C(═O)(C₁₋₄alkylene)N(C₁₋₄alkyl)₂,C₃₋₇cycloalkyl, phenyl, benzyl, phenylethyl, phenyloxy, benzyloxy,naphthyl, a 4- to 7-membered heterocyclo, and/or a 5- to 6-memberedheteroaryl. When a substituted alkyl is substituted with an aryl,heterocyclo, cycloalkyl, or heteroaryl group, said ringed systems are asdefined below and thus may have 0, 1, 2, or 3 substituents, also asdefined below.

When the term “alkyl” is used together with another group, such as in“arylalkyl”, this conjunction defines with more specificity at least oneof the substituents that the substituted alkyl will contain. Forexample, “arylalkyl” refers to a substituted alkyl group as definedabove where at least one of the substituents is an aryl, such as benzyl.Thus, the term aryl(C₀₋₄)alkyl includes a substituted lower alkyl havingat least one aryl substituent and also includes an aryl directly bondedto another group, i.e., aryl(C₀)alkyl.

The term “alkenyl” refers to straight or branched chain hydrocarbongroups having 2 to 12 carbon atoms and at least one double bond. Alkenylgroups of 2 to 6 carbon atoms and having one double bond are mostpreferred. Alkenyl groups may be substituted as described above foralkyl groups.

The term “alkynyl” refers to straight or branched chain hydrocarbongroups having 2 to 12 carbon atoms and at least one triple bond. Alkynylgroups of 2 to 6 carbon atoms and having one triple bond are mostpreferred. Alkynyl groups may be substituted as described above foralkyl groups.

The term “alkylene” refers to bivalent straight or branched chainhydrocarbon groups having 1 to 12 carbon atoms, preferably 1 to 8 carbonatoms, e.g., {—CH₂—}_(n), wherein n is 1 to 12, preferably 1 to 8. Loweralkylene groups, that is, alkylene groups of 1 to 2 carbon atoms, aremost preferred. The terms “alkenylene” and “alkynylene” refer tobivalent radicals of alkenyl and alkynyl groups, respectively, asdefined above. Alkenylene groups may be substituted as described abovefor alkyl groups.

The term “alkoxy” refers to an oxygen atom substituted by alkyl, asdefined herein. For example, the term “alkoxy” or includes the group—O—C₁₋₆alkyl.

When a subscript is used with reference to an alkoxy, thioalkyl oraminoalkyl, the subscript refers to the number of carbon atoms that thegroup may contain in addition to heteroatoms.

It should be understood that the selections for all groups, includingfor examples, alkoxy, thioalkyl, and aminoalkyl, will be made by oneskilled in the field to provide stable compounds.

The term “carbonyl” refers to a bivalent carbonyl group —C(═O)—.

The term “acyl” refers to a carbonyl group linked to an organic radical,more particularly, the group C(═O)R_(e), as well as the bivalent group—C(═O)R_(e)—, which are linked to organic radicals. The group R_(e) canbe selected from alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,or heteroaryl as defined herein, or when appropriate, the correspondingbivalent group, e.g., alkylene.

The term “cycloalkyl” refers to fully saturated and partiallyunsaturated hydrocarbon rings (and therefore includes “cycloalkenylrings”) of 3 to 9, preferably 3 to 7 carbon atoms. The term “cycloalkyl”includes such rings having 0, 1, 2, or 3 substituents selected from(C₁₋₄)alkyl, (C₂₋₄)alkenyl, halogen, hydroxy, cyano, nitro, CF₃,O(C₁₋₄alkyl), OCF₃, C(═O)H, C(═O)(C₁₋₄alkyl), CO₂H, CO₂(C₁₋₄alkyl),NHCO₂(C₁₋₄alkyl), S(C₁₋₄alkyl), NH₂, NH(C₁₋₄alkyl), N(C₁₋₄alkyl)₂,N(C₁₋₄alkyl)₃ ⁺, SO₂(C₁₋₄alkyl), C(═O)(C₁₋₄alkylene)NH₂,C(═O)(C₁₋₄alkylene)NH(alkyl), and/or C(═O)(C₁₋₄alkylene)N(C₁₋₄alkyl)₂.The term “cycloalkyl” also includes such rings having a second ringfused thereto (e.g., including benzo, heterocyclo, or heteroaryl rings)or having a carbon-carbon bridge of 3 to 4 carbon atoms.

The term “halo” or “halogen” refers to chloro, bromo, fluoro and iodo.

The term “haloalkyl” means a substituted alkyl having one or more halosubstituents. For example, “haloalkyl” includes mono, bi, andtrifluoromethyl.

The term “haloalkoxy” means an alkoxy group having one or more halosubstituents. For example, “haloalkoxy” includes OCF₃.

The term “heteroatoms” shall include oxygen, sulfur and nitrogen.

The term “aryl” refers to phenyl, biphenyl, fluorenyl, 1-naphthyl and2-naphthyl. The term “aryl” includes such rings having 0, 1, 2 or 3substituents selected from (C₁₋₄)alkyl, (C₂₋₄)alkenyl, halogen, hydroxy,cyano, nitro, CF₃, O(C₁₋₄alkyl), OCF₃, C(═O)H, C(═O)(C₁₋₄alkyl), CO₂H,CO₂(C₁₋₄alkyl), NHCO₂(C₁₋₄alkyl), S(C₁₋₄alkyl), NH₂, NH(C₁₋₄alkyl),N(C₁₋₄alkyl)₂, N(C₁₋₄alkyl)₃ ⁺, SO₂(C₁₋₄alkyl), C(═O)(C₁₋₄alkylene)NH₂,C(═O)(C₁₋₄alkylene)NH(alkyl), and/or C(═O)(C₁₋₄alkylene)N(C₁₋₄alkyl)₂.

The terms “heterocyclo” or “heterocyclic” refers to substituted andunsubstituted non-aromatic (which may be partially or fully saturated)3- to 15-membered rings having 1 to 4 heteroatoms. Such rings can be 3-to 7-membered monocyclic groups, 7- to 11-membered bicyclic groups, and10- to 15-membered tricyclic groups. Each ring of the heterocyclo groupcontaining a heteroatom can contain one or two oxygen or sulfur atomsand/or from 1 to 4 nitrogen atoms provided that the total number ofheteroatoms in each ring is four or less, and further provided that thering contains at least one carbon atom. The fused rings completingbicyclic and tricyclic groups may contain only carbon atoms and may besaturated, partially saturated, or unsaturated. The nitrogen and sulfuratoms may optionally be oxidized and the nitrogen atoms may optionallybe quaternized. The heterocyclo group may be attached at any availablenitrogen or carbon atom. The heterocyclo ring may contain 0, 1, 2 or 3substituents selected from (C₁₋₄)alkyl, (C₂₋₄)alkenyl, halogen, hydroxy,cyano, nitro, CF₃, O(C₁₋₄alkyl), OCF₃, C(═O)H, C(═O)(C₁₋₄alkyl), CO₂H,CO₂(C₁₋₄alkyl), NHCO₂(C₁₋₄alkyl), S(C₁₋₄alkyl), NH₂, NH(C₁₋₄alkyl),N(C₁₋₄alkyl)₂, N(C₁₋₄alkyl)₃ ⁺, SO₂(C₁₋₄alkyl), C(═O)(C₁₋₄alkylene)NH₂,C(═O)(C₁₋₄alkylene)NH(alkyl), and/or C(═O)(C₁₋₄alkylene)N(C₁₋₄alkyl)₂.Exemplary heterocyclic groups include azetidinyl, pyrrolidinyl,oxetanyl, imidazolinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl,isothiazolidinyl, tetrahydrofuranyl, piperidyl, piperazinyl,2-oxopiperazinyl, 2-oxopiperidyl, 2-oxopyrrolodinyl, 2-oxoazepinyl,azepinyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl,thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone,1,3-dioxolane, quinuclidinyl, tetrahydro-1,1-dioxothienyl and the like.

The term “heteroaryl” refers to substituted and unsubstituted aromatic3- to 14-membered rings having 1 to 4 heteroatoms selected from O, S, orN in at least one of the rings. Said rings can be 5- or 6-memberedmonocyclic groups, 9- or 10-membered bicyclic groups, and 11- to14-membered tricyclic groups. Each ring of the heteroaryl groupcontaining a heteroatom can contain one or two oxygen or sulfur atomsand/or from 1 to 4 nitrogen atoms provided that the total number ofheteroatoms in each ring is four or less and each ring has at least onecarbon atom. The fused rings completing the bicyclic and tricyclicgroups may contain only carbon atoms and may be saturated, partiallysaturated, or unsaturated. The nitrogen and sulfur atoms may optionallybe oxidized and the nitrogen atoms may optionally be quaternized.Heteroaryl groups which are bicyclic or tricyclic must include at leastone fully aromatic ring but the other fused ring or rings may bearomatic or non-aromatic. The heteroaryl group may be attached at anyavailable nitrogen or carbon atom of any ring. The heteroaryl ringsystem may contain 0, 1, 2 or 3 substituents selected from (C₁₋₄)alkyl,(C₂₋₄)alkenyl, halogen, hydroxy, cyano, nitro, CF₃, O(C₁₋₄alkyl), OCF₃,C(═O)H, C(═O)(C₁₋₄alkyl), CO₂H, CO₂(C₁₋₄alkyl), NHCO₂(C₁₋₄alkyl),S(C₁₋₄alkyl), NH₂, NH(C₁₋₄alkyl), N(C₁₋₄alkyl)₂, N(C₁₋₄alkyl)₃ ⁺,SO₂(C₁₋₄alkyl), C(═O)(C₁₋₄alkylene)NH₂, C(═O)(C₁₋₄alkylene)NH(alkyl),and/or C(═O)(C₁₋₄alkylene)N(C₁₋₄alkyl)₂.

Exemplary heteroaryl groups include pyrrolyl, pyrazolyl, pyrazolinyl,imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl,furanyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl,pyridazinyl, triazinyl, indolyl, benzothiazolyl, benzodioxolyl,benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl,isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl,chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl,indazolyl, pyrrolopyridyl, furopyridyl, dihydroisoindolyl,tetrahydroquinolinyl and the like. Particular heteroaryl groups include,for example, 6-substituted quinazolin-4-yl and6-trifluoromethyl-quinazolin-4-yl.

Where a group is optionally substituted, it shall include substitutedand unsubstituted groups.

The compounds herein described may have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom maybe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Many geometric isomers of olefins, C═N double bonds, and the like canalso be present in the compounds described herein, and all such stableisomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral, diastereomeric, racemic forms and allgeometric isomeric forms of a structure are intended, unless thespecific stereochemistry or isomeric form is specifically indicated.

One enantiomer of compounds disclosed herein may display superioractivity compared with the other. Thus, all of the stereochemistries areconsidered to be a part of the present invention. When required,separation of the racemic material can be achieved by HPLC using achiral column or by a resolution using a resolving agent such ascamphonic chloride as in Young, S. D. et al., Antimicrobial Agents andChemotherapy, 2602-2605 (1995).

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, benzene sulfonic, hydrobromic, sulfuric,sulfamic, phosphoric, nitric and the like; and the salts prepared fromorganic acids such as acetic, propionic, succinic, glycolic, stearic,lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,p. 1418 (1985), the disclosure of which is hereby incorporated byreference.

Since prodrugs are known to enhance numerous desirable qualities ofpharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.)the compounds of the present invention may be delivered in prodrug form.Thus, the present invention is intended to cover prodrugs of thepresently claimed compounds, methods of delivering the same andcompositions containing the same. “Prodrugs” are intended to include anycovalently bonded carriers which release an active parent drug of thepresent invention in vivo when such prodrug is administered to amammalian subject. Prodrugs in the present invention are prepared bymodifying functional groups present in the compound in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compound. Prodrugs include compounds of the presentinvention wherein a hydroxy, amino, or sulfhydryl group is bonded to anygroup that, when the prodrug of the present invention is administered toa mammalian subject, it cleaves to form a free hydroxyl, free amino, orfree sulfhydryl group, respectively. Examples of prodrugs include, butare not limited to, acetate, formate and benzoate derivatives of alcoholand amine functional groups in the compounds of the present invention.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent. The present invention is intended toembody stable compounds.

“Therapeutically effective amount” is intended to include an amount of acompound of the present invention alone or an amount of the combinationof compounds claimed or an amount of a compound of the present inventionin combination with other active ingredients effective to inhibit bothCCR-2 and CCR-5 or effective to treat or prevent disorders as discussedherein.

As used herein, “treating” or “treatment” cover the treatment of adisease-state in a mammal, particularly in a human, and include: (a)preventing the disease-state from occurring in a mammal, in particular,when such mammal is predisposed to the disease-state but has not yetbeen diagnosed as having it; (b) inhibiting the disease-state, i.e.,arresting it development; and/or (c) relieving the disease-state, i.e.,causing regression of the disease state.

The names used herein to designate a specific form, e.g., “N-1” or“P-1”, should not be considered limiting with respect to any othersubstance possessing similar or identical physical and chemicalcharacteristics, but rather it should be understood that thesedesignations are mere identifiers that should be interpreted accordingto the characterization information also presented herein.

The present invention provides crystalline forms of the free base ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamideas a novel material, in particular, in a pharmaceutically acceptableform. In certain preferred embodiments, crystalline forms of the freebase are in substantially pure form. Preferred embodiments of the freebase are disclosed in the examples as the N-1 form. Additionally, it isbelieved that the free base may exist as the P-1 form and/or acombination of the N-1 and P-1 forms.

As used herein “polymorph” refers to crystalline forms having the samechemical composition but different spatial arrangements of themolecules, atoms, and/or ions forming the crystal.

As used herein “solvate” refers to a crystalline form of a molecule,atom, and/or ions that further contains molecules of a solvent orsolvents incorporated into the crystalline structure. The solventmolecules in the solvate may be present in a regular arrangement and/ora non-ordered arrangement. The solvate may comprise either astoichiometric or nonstoichiometric amount of the solvent molecules. Forexample, a solvate with a nonstoichiometric amount of solvent moleculesmay result from partial loss of solvent from the solvate.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include deuteriumand tritium. Isotopes of carbon include ¹³C and ¹⁴C.Isotopically-labeled compounds of the invention can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described herein, using an appropriateisotopically-labeled reagent in place of the non-labeled reagentotherwise employed.

As used herein “amorphous” refers to a solid form of a molecule, atom,and/or ions that is not crystalline. An amorphous solid does not displaya definitive x-ray diffraction pattern.

As used herein, “substantially pure,” when used in reference to acrystalline form, means a compound having a purity greater than 90weight %, including greater than 90, 91, 92, 93, 94, 95, 96, 97, 98 and99 weight %, and also including equal to about 100 weight % of CompoundI, based on the weight of the compound. The remaining material comprisesother form(s) of the compound, and/or reaction impurities and/orprocessing impurities arising from its preparation. For example, acrystalline form ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,free base or salt, may be deemed substantially pure in that it has apurity greater than 90 weight %, as measured by means that are at thistime known and generally accepted in the art, where the remaining lessthan 10 weight % of material comprises other form(s) ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,free base or salt, and/or reaction impurities and/or processingimpurities.

Samples of the crystalline forms may be provided with substantially purephase homogeneity, indicating the presence of a dominant amount of asingle crystalline form and optionally minor amounts of one or moreother crystalline forms. The presence of more than one crystalline formin a sample may be determined by techniques such as powder x-raydiffraction (PXRD) or solid state nuclear magnetic resonancespectroscopy (SSNMR). For example, the presence of extra peaks in thecomparison of an experimentally measured PXRD pattern with a simulatedPXRD pattern may indicate more than one crystalline form in the sample.The simulated PXRD may be calculated from single crystal x-ray data. SeeSmith, D. K., “A FORTRAN Program for Calculating X-Ray PowderDiffraction Patterns,” Lawrence Radiation Laboratory, Livermore, Calif.,UCRL-7196 (April 1963).

Preferably, the crystalline form has substantially pure phasehomogeneity as indicated by less than 10%, preferably less than 5%, andmore preferably less than 2% of the total peak area in theexperimentally measured PXRD pattern arising from the extra peaks thatare absent from the simulated PXRD pattern. Most preferred is acrystalline form having substantially pure phase homogeneity with lessthan 1% of the total peak area in the experimentally measured PXRDpattern arising from the extra peaks that are absent from the simulatedPXRD pattern.

Procedures for the preparation of crystalline forms are known in theart. The crystalline forms may be prepared by a variety of methods,including for example, crystallization or recrystallization from asuitable solvent, sublimation, growth from a melt, solid statetransformation from another phase, crystallization from a supercriticalfluid, and jet spraying. Techniques for crystallization orrecrystallization of crystalline forms from a solvent mixture include,for example, evaporation of the solvent, decreasing the temperature ofthe solvent mixture, crystal seeding a supersaturated solvent mixture ofthe molecule and/or salt, freeze drying the solvent mixture, andaddition of antisolvents (countersolvents) to the solvent mixture.

The forms may be characterized and distinguished using single crystalx-ray diffraction, which is based on unit cell measurements of a singlecrystal of a form at a fixed analytical temperature. A detaileddescription of unit cells is provided in Stout et al., Chapter 3, X-RayStructure Determination: A Practical Guide, Macmillan Co., New York(1968). Alternatively, the unique arrangement of atoms in spatialrelation within the crystalline lattice may be characterized accordingto the observed fractional atomic coordinates. Another means ofcharacterizing the crystalline structure is by powder x-ray diffractionanalysis in which the experimental or observed diffraction profile iscompared to a simulated profile representing pure powder material, bothrun at the same analytical temperature, and measurements for the subjectform characterized as a series of 2θ values.

Other means of characterizing the form may be used, such as solid statenuclear magnetic resonance (SSNMR), differential scanning calorimetryand thermogravimetric analysis. These parameters may also be used incombination to characterize the subject form.

The term “negligible weight loss,” as employed herein, as characterizedby TGA indicates the presence of a neat (non-solvated) crystal form.

In one embodiment of the invention, a crystalline form ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,free base or salt, is provided in substantially pure form. Thiscrystalline form may be employed in pharmaceutical compositions whichmay optionally include one or more other components selected, forexample, from the group consisting of excipients, carriers, and one ofother active pharmaceutical ingredients or active chemical entities ofdifferent molecular structures.

Preferably, the crystalline form has substantially pure phasehomogeneity as indicated by less than 10%, preferably less than 5%, andmore preferably less than 2% of the total peak area in theexperimentally measured PXRD pattern arising from the extra peaks thatare absent from the simulated PXRD pattern. Most preferred is acrystalline form having substantially pure phase homogeneity with lessthan 1% of the total peak area in the experimentally measured PXRDpattern arising from the extra peaks that are absent from the simulatedPXRD pattern.

In another embodiment, a composition is provided consisting essentiallyof the crystalline forms ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,free base or salt. The composition of this embodiment may comprise atleast 90 weight % of the form, based on its weight in the composition.

The presence of reaction impurities and/or processing impurities may bedetermined by analytical techniques known in the art, such as, forexample, chromatography, nuclear magnetic resonance spectroscopy, massspectrometry or infrared spectroscopy.

Crystalline forms may be prepared by a variety of methods, including forexample, crystallization or recrystallization from a suitable solvent,sublimation, growth from a melt, solid state transformation from anotherphase, crystallization from a supercritical fluid, and jet spraying.Techniques for crystallization or recrystallization of crystalline formsfrom a solvent mixture include, for example, evaporation of the solvent,decreasing the temperature of the solvent mixture, crystal seeding asupersaturated solvent mixture of the molecule and/or salt, freezedrying the solvent mixture, and addition of antisolvents(countersolvents) to the solvent mixture. High throughputcrystallization techniques may be employed to prepare crystalline formsincluding polymorphs.

Crystals of drugs, including polymorphs, methods of preparation, andcharacterization of drug crystals are discussed in Byrn, S. R. et al.,Solid-State Chemistry of Drugs, 2nd Edition, SSCI, West Lafayette, Ind.(1999).

For crystallization techniques that employ solvent, the choice ofsolvent or solvents is typically dependent upon one or more factors,such as solubility of the compound, crystallization technique, and vaporpressure of the solvent. Combinations of solvents may be employed; forexample, the compound may be solubilized into a first solvent to afforda solution, followed by the addition of an antisolvent to decrease thesolubility of the compound in the solution and to afford the formationof crystals. An “antisolvent” is a solvent in which the compound has lowsolubility. Suitable solvents for preparing crystals include polar andnonpolar solvents.

In one method to prepare crystals,N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,free base or salt, is suspended and/or stirred in a suitable solvent toafford a slurry, which may be heated to promote dissolution. The term“slurry,” as used herein, means a saturated solution, which may alsocontain an additional amount of the solid to afford a heterogeneousmixture at a given temperature. Suitable solvents in this regardinclude, for example, polar aprotic solvents and polar protic solvents,and mixtures of two or more of these, as disclosed herein.

Seed crystals may be added to any crystallization mixture to promotecrystallization. As will be clear to the skilled artisan, seeding isused as a means of controlling growth of a particular crystalline formor as a means of controlling the particle size distribution of thecrystalline product. Accordingly, calculation of the amount of seedsneeded depends on the size of the seed available and the desired size ofan average product particle as described, for example, in Mullin, J. W.et al., “Programmed cooling of batch crystallizers,” ChemicalEngineering Science, 26:369-377 (1971). In general, seeds of small sizeare needed to effectively control the growth of crystals in the batch.Seeds of small size may be generated by sieving, milling, or micronizingof larger crystals, or by micro-crystallization of solutions. Careshould be taken that milling or micronizing of crystals does not resultin any change in crystallinity from the desired crystal form (i.e.,change to amorphous or to another polymorph).

A cooled mixture may be filtered under vacuum, and the isolated solidsmay be washed with a suitable solvent, such as cold recrystallizationsolvent, and dried under a nitrogen purge to afford the desiredcrystalline form. The isolated solids may be analyzed by a suitablespectroscopic or analytical technique, such as SSNMR, DSC, PXRD, or thelike, to assure formation of the preferred crystalline form of theproduct. The resulting crystalline form is typically produced in anamount of greater than about 70 weight % isolated yield, but preferablygreater than 90 weight % based on the weight ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,free base or salt, originally employed in the crystallization procedure.The product may be co-milled or passed through a mesh screen to de-lumpthe product, if necessary.

Crystalline forms may be prepared directly from the reaction medium ofthe final process step for preparingN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,free base or salt. This may be achieved, for example, by employing inthe final process step a solvent or mixture of solvents from which thecompound may be crystallized. Alternatively, crystalline forms may beobtained by distillation or solvent addition techniques. Suitablesolvents for this purpose include any of those solvents describedherein, including protic polar solvents, such as alcohols, and aproticpolar solvents, such as ketones.

By way of general guidance, the reaction mixture may be filtered toremove any undesired impurities, inorganic salts, and the like, followedby washing with reaction or crystallization solvent. The resultingsolution may be concentrated to remove excess solvent or gaseousconstituents. If distillation is employed, the ultimate amount ofdistillate collected may vary, depending on process factors including,for example, vessel size, stirring capability, and the like. By way ofgeneral guidance, the reaction solution may be distilled to about 1/10the original volume before solvent replacement is carried out. Thereaction may be sampled and assayed to determine the extent of thereaction and the wt % product in accordance with standard processtechniques. If desired, additional reaction solvent may be added orremoved to optimize reaction concentration. Preferably, the finalconcentration is adjusted to about 50 wt % at which point a slurrytypically results.

It may be preferable to add solvents directly to the reaction vesselwithout distilling the reaction mixture. Preferred solvents for thispurpose are those which may ultimately participate in the crystallinelattice, as discussed above in connection with solvent exchange.Although the final concentration may vary depending on desired purity,recovery and the like, the final concentration of the free base insolution is preferably about 4% to about 7%. The reaction mixture may bestirred following solvent addition and simultaneously warmed. By way ofillustration, the reaction mixture may be stirred for about 1 hour whilewarming to about 70° C. The reaction is preferably filtered hot andwashed with either the reaction solvent, the solvent added or acombination thereof. Seed crystals may be added to any crystallizationsolution to initiate crystallization.

The various forms described herein may be distinguishable from oneanother through the use of various analytical techniques known to one ofordinary skill in the art. Such techniques include, but are not limitedto, powder x-ray diffraction (PXRD), differential scanning calorimetry(DSC) and/or thermogravimetric analysis (TGA). Alternatively, the formsmay be characterized and distinguished using single crystal x-raydiffraction, which is based on unit cell measurements of a singlecrystal of a given form at a fixed analytical temperature. A detaileddescription of unit cells is provided in Stout et al., Chapter 3, X-RayStructure Determination: A Practical Guide, Macmillan Co., New York(1968). Specifically, the unique arrangement of atoms in spatialrelation within the crystalline lattice may be characterized accordingto the observed fractional atomic coordinates. Another means ofcharacterizing the crystalline structure is by powder x-ray diffractionanalysis in which the observed diffraction profile is compared to asimulated profile generated from single crystal structure data. Powderx-ray diffraction measurements for the subject form are characterized asa series of 2θ values (usually four or more).

Other means of characterizing the form may be used, such as solid statenuclear magnetic resonance (SSNMR) spectroscopy, differential scanningcalorimetry (DSC), thermography and gross examination of the crystallineor amorphous morphology. These parameters may also be used incombination to characterize the subject form.

One of ordinary skill in the art will appreciate that an x-raydiffraction pattern may be obtained with a measurement error that isdependent upon the measurement conditions employed. In particular, it isgenerally known that intensities in an x-ray diffraction pattern mayfluctuate depending upon measurement conditions employed and the shapeor morphology of the crystal. It should be further understood thatrelative intensities may also vary depending upon experimentalconditions and, accordingly, the exact order of intensity should not betaken into account. Additionally, a measurement error of diffractionangle for a conventional x-ray diffraction pattern is typically about0.2° 2θ values or less, preferably about 0.1° values (as discussedhereinafter), and such degree of measurement error should be taken intoaccount as pertaining to the aforementioned diffraction angles.Consequently, it is to be understood that the crystal forms of theinstant invention are not limited to the crystal forms that providex-ray diffraction patterns completely identical to the x-ray diffractionpatterns depicted in the accompanying Figures disclosed herein. Anycrystal forms that provide x-ray diffraction patterns substantiallyidentical to those disclosed in the accompanying Figures fall within thescope of the present invention. The ability to ascertain substantialidentities of x-ray diffraction patterns is within the purview of one ofordinary skill in the art.

Synthesis

A carbazide of formula I-ff, such as semicarbazide hydrochloride, wascondensed with a compound of formula I-ee, such as pivaloylacetonitrile, in a solvent, for example, ethanol, 2-propanol, 1-propanoland methanol, preferably ethanol, in the presence of a base, forexample, sodium acetate, potassium acetate, triethylamine andN—N-diisopropyl-N-ethyl amine, preferably sodium acetate, at atemperature in the range of 20-60° C., preferably, in the range of25-55° C., to provide the hydrazone compound of formula I-dd, whereinR₂₁ is ═O; R₂₂ is —NH—NH₂; and R₂₃ is cyanoalkyl; or R₂₂ and R₂₃ may betaken together to form a 5- to 8-membered ring, wherein the ring may beoptionally substituted with one or more substituents selected fromamino, alkyl, aryl, or heteroaryl and may optionally contain 1, 2, 3 or4 heteroatoms independently selected from the group consisting of N, S,or O.

A hydrazone compound of formula I-dd, such as

was reacted with an orthoformate, for example, trimethylorthoformate,triethylorthoformate, and tripropylorthoformate, preferably,trimethylorthoformate, orthoformate in the presence of an acid, forexample, acetic acid, trifluoroacetic acid, hydrochloric acid andmethanesulfonic acid, preferably acetic acid, at elevated temperature,such as 40-90° C., preferably, 40-75° C., to yield the hydrazonecompound of formula I-cc, wherein R₂₁═O and R₂₂ and R₂₃ are as set forthabove.

A hydrazone compound of formula I-cc is converted to a compound ofFormula I-bb, wherein LG is —OR₁₆, wherein R₁₆ is C₁₋₆alkyl, phenyl, a5- to 7-membered heteroaryl having one or more atoms selected from N, S,or O, or a 3- to 7-membered cycloalkyl, all of which are optionallysubstituted with 1 to 3 groups selected from halogen, CF₃ or C₁₋₆alkyl,by reacting the hydrazone compound of formula I-cc, for example,7-tert-butyl-3H-pyrazolo[1,5-a][1,3,5]triazin-4-one, with an acylatingagent, a nucleophile, such as dimethyl 4-aminopyridine (DMAP), and/or atertiary amine base, in a solvent, for example, acetonitrile,dichloromethane and neat phosphorus oxychloride, followed byinterception with either (i) an alcohol in the presence of a secondtertiary amine base or (ii) an alkoxide to form the compound of formulaI-bb. Examples of acylating agents that may be used are phosphorusoxychloride (POCl₃), oxalyl chloride, thionyl chloride and phosgene. Apreferred acylating agent is POCl₃. Triethylamine,N—N-diisopropyl-N-ethyl amine (DIEA), tri-n-propylamine, N-methylmorpholine and 1,4-diazabicyclo[2.2.2]octane (DABCO) are examples oftertiary amine bases that may be used in the conversion. A preferredtertiary amine base is DIEA. Generally, the amount of nucleophile usedin the reaction is 2.0 to 4.0, preferably, 3.0, equivalents. Examples ofalcohols or alkoxides that may be used in the conversion are phenol,pentafluorophenol, methanol, ethanol, sodium methoxide and sodiumphenolate with phenol being the preferred alcohol. Generally, thereaction and interception may be carried out at a temperature in therange of ambient temperature to 70° C.

A compound of formula I-bb, for example,

is coupled with a compound of formula I-aa, prepared in a similar manneras described in WO 2008/014381 A1, wherein: R₁ is independently hydrogenor an amine-protecting group selected from a carbobenzyloxy (Cbz) group,a tert-butyloxycarbonyl (BOC), a fluorenylmethyloxycarbonyl (FMOC)group, a benzyl (Bn) group, and a p-methoxybenzyl (PMB) group,preferably hydrogen; R₈ and R₉ are independently hydrogen or C₁₋₆alkyl;R₂₁ is ═O; HET is an optionally substituted 3- to 14-memberedheterocyclo or heteroaryl bicyclic ring having at least one nitrogenheteroatom, preferably two to four total heteroatoms, especially fournitrogen atoms; and LG is —OR₁₆, wherein R₁₆ is C₁₋₆alkyl, phenyl, a 5-to 7-membered heteroaryl having one or more atoms selected from N, S, orO, or a 3- to 7-membered cycloalkyl, all of which are optionallysubstituted with 1 to 3 groups selected from halogen, CF₃ or C₁₋₆alkyl,to obtain the corresponding compound of formula I, for example,N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,or a salt thereof. Coupling may be performed by coupling methodscommonly known in art, or, alternatively, via an acidic work-up followedby the addition of a base. Examples of acids that may be used in theacidic work-up are citric acid, tartaric acid, glycolic acid, andhydrochloric acid. Examples of bases that may be added are K₂HPO₄,Na₂HPO₄, NaHCO₃ and KHCO₃.

Specifically,N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamidemay be prepared according to the following scheme:

For the process of this invention, starting materials are commerciallyavailable or can be readily prepared by one or ordinary skill in theart. Solvents, temperatures, pressures, starting materials having thedesired groups, and other reaction conditions, may be readily selectedas appropriate by one of ordinary skill in the art. The process can bescaled up in order to prepare larger quantities of the compound offormula I, such as in a commercial production facility.

EXAMPLES

The following Examples illustrate embodiments of the inventive compoundsand starting materials, and are not intended to limit the scope of theclaims.

As appropriate, reactions were conducted under an atmosphere of drynitrogen (or argon). For anhydrous reactions, DRISOLV® solvents from EMwere employed. For other reactions, reagent grade or HPLC grade solventswere utilized. Unless otherwise stated, all commercially obtainedreagents were used as received.

LC/MS measurements were obtained using a Shimadzu HPLC/Waters ZQ singlequadropole mass spectrometer hybrid system. Data for the peak ofinterest are reported from positive-mode electrospray ionization. NMR(nuclear magnetic resonance) spectra were typically obtained on Brukeror JEOL 400 MHz and 600 MHz instruments in the indicated solvents. Allchemical shifts are reported in ppm from tetramethylsilane with thesolvent resonance as the internal standard. ¹H NMR spectral data aretypically reported as follows: chemical shift, multiplicity (s=singlet,br s=broad singlet, d=doublet, dd=doublet of doublets, t=triplet,q=quartet, sep=septet, m=multiplet, app=apparent), coupling constants(Hz), and integration.

One of skill in the art will recognize the standard abbreviationsutilized herein. For ease of reference, the abbreviations include, butare not necessarily limited to: Hg=mercury; sat.=saturated,HPLC=high-performance liquid chromatography, AP=area percent,KF=Karl-Fischer, RT=room temperature (unless specified otherwise RT is atemperature of about 22° C.), mmol=millimoles, HRMS=high-resolution massspectroscopy, ° C.=degrees Celsius, kg—kilogram or kilograms, g=gram orgrams, mg=milligram or milligrams, L=liter or liters, mL (orml)=milliliter or milliliters, h or hr=hour or hours, M=molar, N=normal,min=minute or minutes, MHz=megahertz, v/v=volume to volume ratio, %w/w=weight/weight percent, wt %=weight percent, nm=nanometer ornanometers, LOD=loss of drying.

“α”, “β”, “R” and “S” are stereochemical designations familiar to thoseskilled in the art.

Example 1N-((1R,2S,5R)-5-(tert-Butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide

Example 1, Step 1: To a 3 L round-bottom flask was added semicarbazidehydrochloride (100.0 g, 0.89 moles), pivaloyl acetonitrile (112.2 g,0.89 moles) and ethanol (1 L) at 22-25° C. Upon completion of addition,the reaction mixture was cooled to 12-15° C. and anhydrous sodiumacetate (73.5 g, 0.89 moles) was added. The addition of the anhydroussodium acetate was endothermic thereby raising the temperature to 22-25°C. The reaction mixture was maintained at 22-25° C. and stirred for60-90 minutes. After this time, the reaction mixture was analyzed byHPLC, which indicated that the formation of the hydrazone intermediatewas complete.

Example 1, Step 2: The reaction mixture was then heated to 68-72° C. andtrimethylorthoformate (475.7 g, 4.48 moles) was added during a 5-10minute period. The reaction mixture was allowed to cool to 40-45° C. andthen acetic acid (53.8 g, 0.89 moles) was added during a 15-20 minuteperiod. Upon completion of addition, the temperature was raised to 70±2°C. during a 20-25 min period. Once at the prescribed temperature, thereaction mixture was stirred for 18-20 hr. At the conclusion of thisperiod, the reaction mixture was analyzed by HPLC, which indicated thatthe formation of the pyrazolo[1,5-a][1,3,5]triazine was complete.

Example 1, Step 3: The pyrazolo[1,5-a][1,3,5]triazine from Example 1,Step 2, was concentrated at 50-55° C. under reduced pressure (˜5-10 mmHg) to yield a residue. Tetrahydrofuran (THF, 2 L) and acetone (2 L)were added to the residue, and the resulting mixture was stirred at50-55° C. for 90 minutes. After this time, the reaction mixture wasfiltered through Buckner funnel to remove the resultant sodium chloride(NaCl) and sodium acetate (NaOAc) precipitates. The resulting filtratewas concentrated to dryness at 50-55° C. under reduced pressure(˜400-450 mm Hg) to yield a residue. The residue was taken up in2-methylorthoformate (2-methyl THF, 450 mL) and the resulting mixturewas stirred at 22-25° C. for two (2) hours. At the conclusion of thisperiod, the reaction mixture was filtered and then spray washed with2-methyl THF (100 mL), followed by tert-butyl methyl ether (MTBE, 200mL). The resulting product was dried at 45-50° C. reduced pressure(−400-450 mm Hg) to provide7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4(3H)-one (107.0 g, 62.1% w/w,HPLC purity: 99.2 AP at 220 nm). Steps 1 to 3 were repeated on a largerscale to produce kilogram quantities of7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4(3H)-one.

Example 1, Step 4: To a glass lined reactor was added7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4(3H)-one (1.0 kg, 5.2 moles),dimethyl 4-aminopyridine (1.27 kg, 10.4 moles), acetonitrile (10 L), anddiisopropylethylamine (0.672 kg, 5.2 mole). The resulting slurry wasstirred at ambient temperature under a nitrogen atmosphere for a periodof no less than 15 minutes until a clear solution was formed. The clearsolution was slowly added to a second glass lined reactor containingacetonitrile (6.0 L) and phosphorus oxychloride (POCl₃, 0.822 kg, 5.3moles). Upon completion of addition, the resulting mixture was stirredat below 35° C. for 2 hours. At the conclusion of this period, thereaction was analyzed by HPLC, which indicated that the reaction wascomplete. Phenol (0.64 kg, 6.8 mole) and diisopropylethylamine (0.87 kg)were added and the resulting reaction mixture was stirred at ambienttemperature for no less than 1 hr. After this time, the reaction mixturewas analyzed by HPLC, which indicated that the reaction was completed.2-Methyl-THF (20 L), followed by water (10 L) were added to the reactionmixture. The organic and aqueous phases were separated and the aqueousphase was discarded. The organic phase was washed with a citricacid-brine solution (5 wt %, 10 L), and the resulting organic andaqueous phases were separated and the aqueous phase was discarded again.The above citric acid-brine solution wash was repeated two more times.Upon completion of the citric acid-brine solution washes, a potassiumphosphoric base solution (K₂HPO₄, 7.5 wt %, 10 L) was added. The organicand aqueous phases were separated and the aqueous phase was discarded.The above K₂HPO₄ solution wash was repeated one more time until pH ˜8.

Example 1, Step 5:N-[2-(3-Amino-2-oxo-pyrrolidin-1-yl)-5-tert-butylamino-cyclohexyl]-acetamide(prepared in a similar manner as described in U.S. Publication No.2008/0027083 A1, 1.05 kg) was added to the basic organic phase fromExample 1, Step 4. Upon completion of addition, the reaction mixture wasstirred at ambient temperature for 16 hr. At the conclusion of thisperiod, the reaction mixture was analyzed by HPLC, which indicated thereaction was complete. Water (20 L) followed by acetic acid (HOAc, 0.406kg) were added to the reaction mixture, and the resulting organic andaqueous layers were separated. The aqueous layer was extracted with2-methyl THF (10 L). The organic layers were combined and HOAc (0.406kg) was added. The resulting mixture was washed with water (10 L) andthe resulting organic and aqueous layers were separated. This aqueouslayer was extracted again with 2-methyl-THF (10 L). The aqueous layerswere combined again and dichloromethane (DCM, 15 L) was added. Sodiumhydroxide (NaOH, 10 N, 1.04 L) was added to adjust the pH to ˜13.0. Uponcompletion of addition, the organic and aqueous layers were separatedagain and the product-rich DCM layer was set aside. The aqueous layerwas extracted with additional dichloromethane (10 L). The DCM richorganic layers were combined and washed with water (10 L). The resultingproduct rich DCM solution was concentrated in vacuo to a minimum volume.Ethyl acetate (EtOAc) was added and the residual DCM and water werecontinuously distilled off to yield a slurry (final volume ˜5 L). MTBE(15 L) was added and the resulting slurry was stirred for no less than 1hr. After this time, the slurry was filter and the wet filter cake waswashed with additional MTBE (5 L). The wet cake was dried at 55° C. invacuo until LOD≦0.5 wt % to afford the amorphous free base of Example 1(0.9 kg, 55 M % yield, HPLC purity: 99.5 AP). ¹H NMR (600.13 MHz,DMSO-d₆) δ 1.04 (s, 9H), 1.34 (s, 9H), 1.58 (m, 3H), 1.64 (m, 2H), 1.81(s, 3H), 2.05 (m, 1H), 2.12 (m, 1H), 2.36 (m, 1H), 2.93 (br s, 1H), 3.48(m, 2H), 3.84 (m, 1H), 4.26 (br s, 1H), 4.86 (t, J=8.9 Hz, 1H), 6.39 (s,1H), 8.07 (s, 1H), 8.94 (br s, 1H); ¹³C NMR (125.8 MHz, DMSO-d₆) δ 21.3,23.3, 25.9, 29.3, 30.0, 32.2, 32.6, 35.5, 43.1, 46.5, 47.7, 50.7, 51.7,52.5, 92.3, 148.7, 148.8, 152.9, 167.3, 168.5, 171.2; HRMS calcd forC₂₅H₄₀N₈O₂ (M+1) 485.3274. found 485.3343.

Example 2 Tritium-labeledN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide

Example 3 Deuterium-labeled N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide

Example 4 Carbon-14 labeledN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide

Example 5 N-1 Crystal Form ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide

A free base crystal form ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,including salt forms, and solvates thereof, was prepared andcharacterized as described below.

Procedures for Characterizing the Forms

Single Crystal Data

Data were collected on a Bruker-Nonius (Bruker AXS, Inc., 5465 EastCheryl Parkway Madison, Wis. 53711 USA) CAD4 serial diffractometer. Unitcell parameters were obtained through least-squares analysis of theexperimental diffractometer settings of 25 high-angle reflections.Intensities were measured using Cu Kα radiation (μ=1.5418 Å) at aconstant temperature with the θ-2θ variable scan technique and werecorrected only for Lorentz-polarization factors. Background counts werecollected at the extremes of the scan for half of the time of the scan.Alternately, single crystal data were collected on a Bruker-Nonius KappaCCD 2000 system using Cu Kα radiation (λ=1.5418 Å). Indexing andprocessing of the measured intensity data were carried out with theHKL2000 software package (Otwinowski, Z. et al. in MacromolecularCrystallography, Carter, W. C., Jr. et al., eds., Academic, NY, publ.,Vol. 276, pp. 307-326 (1997)) in the Collect program suite. (CollectData collection and processing user interface: Collect: Data collectionsoftware, R. Hooft, Nonius B. V., 1998.) Alternately, single crystaldata were collected on a Bruker-AXS APEX2 CCD system using Cu Kαradiation (λ=1.5418 Å). Indexing and processing of the measuredintensity data were carried out with the APEX2 software package/programsuite (APEX2 Data collection and processing user interface: APEX2 UserManual, v1.27; Bruker AXS, Inc., 5465 East Cheryl Parkway Madison, Wis.53711 USA).

When indicated, crystals were cooled in the cold stream of an Oxfordcryo system (Oxford Cryosystems Cryostream cooler: Cosier, J. et al., J.Appl. Cryst., 19:105 (1986)) during data collection.

The structures were solved by direct methods and refined on the basis ofobserved reflections using either the SDP (SDP, Structure DeterminationPackage, Enraf-Nonius, Bohemia N.Y. 11716. Scattering factors, includingf′ and f″, in the SDP software were taken from the International Tablesfor Crystallography, Kynoch Press, Birmingham, England, Vol. IV, Tables2.2A and 2.3.1 (1974)) software package with minor local modificationsor the crystallographic packages MAXUS (maXus solution and refinementsoftware suite: Mackay, S. et al., maXus: a computer program for thesolution and refinement of crystal structures from diffraction data orSHELXTL4. The derived atomic parameters (coordinates and temperaturefactors) were refined through full matrix least-squares. The functionminimized in the refinements was Σ_(w)(|F_(o)|−|F_(c)|)². R is definedas Σ∥F_(o)|−|F_(c)∥/ρ|F_(o)| whileR_(w)=[Σ_(w)(|F_(o)|−|F_(c)|)²/Σ_(w)|F_(o)|²]^(1/2) where w is anappropriate weighting function based on errors in the observedintensities. Difference maps were examined at all stages of refinement.Hydrogens were introduced in idealized positions with isotropictemperature factors, but no hydrogen parameters were varied.

X-Ray Powder Diffraction Data (PXRD)

PXRD data were obtained using a Bruker C2 GADDS. The radiation was Cu Kα(40 KV, 50 mA). The sample-detector distance was 15 cm. Powder sampleswere placed in sealed glass capillaries of 1 mm or less in diameter; thecapillary was rotated during data collection. Data were collected for3≦2θ≦35° with a sample exposure time of at least 2000 seconds. Theresulting two-dimensional diffraction arcs were integrated to create atraditional 1-dimensional PXRD pattern with a step size of 0.02 degrees2θ in the range of 3 to 35 degrees 2θ. About 200 mg were packed into aPhilips powder x-ray diffraction (PXRD) sample holder. The sample wastransferred to a Philips MPD unit (45 KV, 40 mA, Cu Kα). Data werecollected at room temperature in the 2 to 32 2-theta range (continuousscanning mode, scanning rate 0.03 degrees/sec., auto divergence and antiscatter slits, receiving slit: 0.2 mm, sample spinner: ON)

Differential Scanning Calorimetry (DSC)

DSC experiments were performed in a TA INSTRUMENTS® model Q1000 or 2920.The sample (about 2-6 mg) was weighed in an aluminum pan and recordedaccurately recorded to a hundredth of a milligram, and transferred tothe DSC. The instrument was purged with nitrogen gas at 50 mL/min. Datawere collected between room temperature and 300° C. at 10° C./minheating rate. The plot was made with the endothermic peaks pointingdown.

Thermal Gravimetric Analysis (TGA)

TGA experiments were performed in a TA INSTRUMENTS® model Q500 or 2950.The sample (about 10-30 mg) was placed in a previously tared platinumpan previously tared. The weight of the sample was measured accuratelyand recorded to a thousandth of a milligram by the instrument. Thefurnace was purged with nitrogen gas at 100 mL/min. Data were collectedbetween room temperature and 300° C. at 10° C./min heating rate.

Preparation and Analysis of the Forms

The unit cell data and other properties for the example are presented inTable 1. The unit cell parameters were obtained from single crystalx-ray crystallographic analysis. A detailed account of unit cells can befound in Chapter 3 of Stout et al., X-Ray Structure Determination: aPractical Guide, Macmillan (1968).

Finally, FIG. 1 presents the XRPD pattern for Example 5. FIGS. 2 and 3disclose the DSC and TGA analysis, respectively, of Example 5.

Form Preparation, XRD, DSC and TGA Characterization Example 5, N-1 Form,Free Base

N-((1R,2S,5R)-5-(tert-Butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide,form N-1, was crystallized from ethyl acetate and MTBE. Form N-1, is aneat (no molecules of water or solvent) form ofN-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide.Form N-1 was characterized by a XRD pattern which matches the simulatedpattern generated from the single crystal structure data. Form N-1 wascharacterized by a DSC thermogram having a melt/decomposition endothermwith an onset typically at ca. 205° C. Form N-1 was characterized by aTGA thermal curve having a weight loss at up to ca. 210° C.

TABLE 1 Unit Cell Parameters Compound Exp 5 Structure Base Form N-1 T−70 a(Å) 7.3085(6) b(Å) 16.257(1) c(Å) 22.688(2) α° 90 β° 90 γ° 90 V(Å³)2695.4(4) Z′ 1 Vm 673 Sg P2₁2₁2₁ Dcalc 1.194

The variables used in Table 1 are defined below:

T=temperature in Centigrade for the crystallographic data (RT is roomtemperature which is about +22° C.)

V=volume of unit cell

Z′=number of drug molecules per asymmetric unit

Vm=V(unit cell)/(Z drug molecules per cell)

sg=space group

dcalc=calculated crystal density

Comparative Pharmacological Characteristics

Assays and data comparing the pharmacological characteristics of Example1 and compounds found in WO 2005/021500 A1, WO 2008/014381 A1 and WO2008/014360 A1 are presented below.

Human Peripheral Blood Mononuclear Cell Binding

See also: Yoshimura et al., J. Immunol., 145:292 (1990). The human CCR-2binding assay was established with human peripheral blood mononuclearcells (hPBMCs) using ¹²⁵I-human MCP-1 as the tracer ligand. hPBMCs wereisolated from human leukopak (Biological Specialty Inc.) using astandard protocol with Ficoll-Hypaque (Mediatech CELLGRO®). IsolatedhPBMCs were washed and diluted to 1×10⁷/ml in binding buffer (RPMI-1640,0.1% BSA, 20 mM Hepes, pH 7.4). ¹²⁵I-MCP-1 (NEN/Perkin Elmer) wasdiluted to 0.45 nM in binding buffer. The compound was diluted inbinding buffer at 3-fold the final concentrations used in the bindingassay. The binding assay was performed using a 96-well filter plate(Millipore). Total ¹²⁵I-MCP-1 binding was assessed as follows: to eachreaction of a total volume of 150 μl were added 5×10⁵ cells, 0.15 nM¹²⁵I-MCP-1, and compound such that the final concentration ranged from 0to 100 nM. The plate was incubated at room temperature for 30 minutesfollowed by three washes with RPMI-1640, 0.1% BSA, 0.4 M NaCl, 20 mMHepes, pH 7.4 using a vacuum manifold filtration (Millipore). Afterwashing, the plate was air-dried for 60 minutes at room temperature.This was followed by adding 25 μl of Microscint 20 into each well. Theplate was sealed and counted on the Trilux for 1 minute. Non-specificbinding was determined in the presence of 300 nM cold MCP-1 (PeproTechInc.). Specific ¹²⁵I-MCP-1 was calculated as the difference betweentotal and non-specific binding. All conditions were tested in duplicate.The IC50 is defined as the concentration of competing compound requiredto reduce specific binding by 50%.

The procedure used for T cell CCR-5 binding assay was similar to thosefor hPBMC CCR-2 binding assay except that human peripheral T cells wasused as a source of CCR-5 (see below) and ¹²⁵I-MIP-1β as a tracer(Amersham).

Isolation of Peripheral T Cells

A recent report indicates that CCR-5 expression on T cells variesconsiderably among individuals (Desmetz, C. et al., “The strength of thechemotactic response to a CCR-5 binding chemokine is determined by thelevel of cell surface CCR-5 density”, Immunology, 119(4):551-561(2006)). Therefore, blood donors were pre-screened for high T-cellexpression of CCR-5. hPBMCs were first isolated from human whole bloodby standard protocol with Ficoll-Hypaque. Flow cytometric analysis wasthen used to measure CCR-5 expression on T cells following staining withanti-CCR-5 antibody plus anti-CD4 antibody or anti-CD8 antibody. Thoseblood donors in which >5% peripheral T cells (CD4⁺ and CD8⁺) expressCCR-5 were requested to provide blood again for isolation of PBMCs and,subsequently, of T cells using a standard E-rosetting technique whichrelies on the unique ability of T cells to bind to sheep red blood cells(RBCs).

CCR-2 Chemotaxis

The human CCR-2 chemotaxis assay was conducted with the human monocyticcell line, THP-1. THP-1 cells were first labeled with the fluorescentdye Calcein-AM in phenol red-free, BSA-free RPMI-1640 (pH 7.4) at 37° C.for 30 minutes with gentle mixing every 15 minutes. The labeled cellswere then washed and re-suspended at 1×10⁵/ml in chemotaxis buffer(phenol red-free RPMI-1640, 0.1% BSA, pH 7.4). The test compound wasdiluted in chemotaxis buffer such that the final assay concentrationranged from 0.01 nM to 1 μM. The ligand MCP-1 (PeproTech Inc.) wasdiluted to 20 nM in chemotaxis buffer. To perform the assay, an equalvolume of test compound dilutions was mixed with an equal volume oflabeled THP-1 cells (Mixture 1), and an equal volume of test compounddilutions was mixed with an equal volume of diluted MCP-1 ligand(Mixture 2). Both mixtures were incubated independently at 37° C. for 10minutes followed by gentle mixing. MCP-1-induced chemotaxis was thenmeasured in a chemotaxis plate (Becton Dickinson) by placing 50 μl ofMixture 1 in the top chamber and 225 μl of Mixture 2 in the bottomchamber. The plate was covered with a lid and incubated at 37° C. forminutes. 30 minutes later, the plate was read on a CYTOFLUOR®. Allconditions were tested in duplicate. For signal to noise determination,50 μl of labeled THP-1 cells alone (5×10⁴/well) were placed into the topchamber and 225 μl of ligand MCP-1 alone was placed in the bottomchamber (final concentration of 10 nM). The inhibition achieved bygraded concentrations of test compound was calculated as a percentage ofthe compound-free MCP-1 control. The IC50 is defined as theconcentration of test compound required to reach 50% inhibition ofcellular chemotaxis.

CCR-5 Chemotaxis

A similar procedure to that set forth above was adopted except thatisolated peripheral T cells was used as CCR-5-expressing cells andMIP-1β (50 nM, PeproTech Inc.) was the ligand.

hERG Flux

HEK293 cells stably-expressing hERG channels were grown (37° C., 5% CO₂)in Dulbecco's Modified Eagle's Media supplemented with 10% Sigma fetalbovine serum, non-essential amino acids, 2 mM L-glutamine and 500 μg/mlG418, at incubator. Cell dissociation buffer was used to extract thecells from flasks, which were then plated into 384-well CORNING®poly-D-lysine coated black/clear plates at a density of 2×10⁴ cells perwell (20 μl) in 10% serum media, and incubated for 15-24 hours at 37° C.in a 5% CO₂ incubator until a confluent monolayer of cells was obtained.

A 2 mM stock of BTC-AM dye (Molecular Probes, Eugene, Oreg.) wasprepared in 100% DMSO and then added 1:1 to 10% (w/v) pluronic acid inDMSO on the day of assay. The dye was then diluted in hERG external EPbuffer (140 mM NaCl, 4.0 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 10 mMHEPES, pH 7.3 and 10 mM glucose; all buffer components obtained fromSigma Chemical). This BTC dye mixture (30 μl) was added to the cells andproduced a final loading concentration of 2.5 μM. Cells are incubated at21° C. for 45 minutes.

The test compound was diluted to 10 mM DMSO in 60 μl. The compound wasthen serially-diluted at a 1:2 ratio in DMSO in columns 1-10 and 11-20of a 384-well plate. Assay-ready plates were generated by stamping 2.5μl from the DMSO serially diluted plate, which was prepared on theVelocity 11 BIOCEL®. Aqueous plates were created by adding 48 μl of EPbuffer and then were diluted 30-45 minutes before the assay was read onthe FLIPR®. After dye loading, aqueous-diluted compound was added to thecells of the three replicate plates (10 μl) yielding a ten pointconcentration range of 80 μM to 0.156 nM. Final DMSO concentration inthe assay is 1%. Assay-ready aqueous plates were prepared and diluted ona CyBio liquid handler.

Cells loaded with dye were read on the FLIPR®384 (Molecular Devices,Sunnyvale, Calif.), which excites the dye using the 488 nm line of anargon laser. Emission was filtered using a 540±30 nm bandpass filter.hERG channels are stimulated to open by the addition of 20 μl/well EPbuffer containing 66 mM K₂SO₄ and 1.3 mM Tl₂SO₄ (Sigma/Aldrich). Foreach plate, data were collected every second for a period of 12 seconds,at which time the Tl⁺-containing stimulus buffer was added. Datacollection proceeded every second for 48 seconds, and then continuedevery three seconds for an additional 2 minutes.

The dynamic range of the assay was determined from blanks and totalswells. The totals wells (columns 21 and 22) define maximal hERGactivation for the plate (no test compound present), and the blankswells (columns 23 and 24) define 100% hERG inhibition. The blanks wellscontain 400 nM of either of the standard hERG inhibitors dofetilide(Ficker et al., 1998) or E-4031. Raw data points in each sample wellwere first corrected for cell/signal variation, negative control(blanks) background, and normalized to the positive controls (totals)using the online FLIPR® software. Test compound concentration responsecurves for the hERG Tl⁺ flux data were then fit using Excel Fit (IDBusiness Solutions Limited, Surrey, UK) with a single-site logisticequation, Y=A+((B−A)/1+((C/X)^D))) where A=maximal inhibition. Data wereanalyzed by fitting maximum amplitudes of change in fluorescence for Tl⁺flux for a given condition of test compound. Potencies (IC₅₀ values) ofcompound were calculated from the average of triplicate wells.

Sodium Channel, Site 2 Binding Assay

See also: Catterall, W. A. et al. J. Biol. Chem., 256:8922 (1981). Thestandard binding buffer contained 50 mM HEPES, 50 mM Tris-HCl, pH 7.4,130 mM choline chloride, 5.4 mM KCl, 0.8 mM MgCl₂, 5.5 mM glucose, 40μg/mL LqT. Binding reactions were initiated by adding synaptosomes(prepared from Wistar rat brain) to the reaction mixture containing 5 nM[³H]-batrachotoxin in a standard binding buffer and the compound to betested at the desirable concentration. Samples were then mixed andincubated at 37° C. for 60 minutes. The reactions were stopped by addingice-cold washing buffer containing 50 mM HEPES, 50 mM Tris-HCl, pH 7.4,1.8 mM CaCl₂, 0.8 mM MgCl₂ and 1 mg/mL bovine serum albumin. Thesynaptosomes were immediately collected onto glass fiber filters andwashed 3 times with washing buffers. The radioactivity of[³H]-batrachotoxin remaining on the filters was counted using liquidscintillation spectrometers.

Parallel Artificial Membrane Permeability Assay (PAMPA)

The Parallel Artificial Membrane Permeability Assay (PAMPA) consists ofa specially formulated lecithin-based lipid combination referred to asthe gastrointestinal tract (GIT) lipid. The GIT lipid is used to form amembrane in a sandwich plate assembly similar to that used in the Caco-2assays. The GIT lipid closely resembles in vivo membrane composition andperformance as measured by standard compounds that are known to bepassively absorbed in humans. PAMPA is widely used as an in vitro modelfor permeability screening of discovery compounds. The rate of passageof compounds through the PAMPA membrane is used to determine apermeability coefficient (Pc), which can be related to the in vivopassive permeability of the compound.

The permeability coefficient (Pc) of a particular compound is examinedin a pH-dependent setting with apical and basolateral pH of 7.4. Allexperiments are conducted in triplicate determinations.

The test compound (10 mM stocks in 100% DMSO) was diluted 1:100 in pH7.4 donor well buffer (pION CAT #110151), providing a 100 μM assaysolution in 1% DMSO. Compound diluted in donor well buffer wastransferred to a Whatman UNIFILTER® plate and filtered prior todispensing 200 μl into the donor well of the assay plate (pION CAT#110163). The PAMPA membrane was formed by pipetting 4 μl of the lipidsolution (pION CAT #110169) onto the filter plate (VWR CAT #13503). Themembrane was then covered with 200 μl of acceptor well buffer at pH 7.4(pION CAT #110139). The PAMPA assay plate (donor side and acceptor side)was combined and allowed to incubate at room temperature for 4 hours.The plate was then disassembled and spectrophotometer plates (VWR CAT#655801) were filled (150 μl/well). The donor, acceptor, reference, andblank plates were read in the SPECTRAMAX® UV plate reader. Data wascaptured by the pION software, which analyzes the spectra and generatesPc values.

hERG Patch Clamp

Whole-cell patch-clamp was used to directly measure hERG currents inHEK-293 cells stably expressing the cloned hERG potassium channel αsubunit. The compound was tested in an aqueous buffer with pH 7.4 atroom temperature. Repetitive test pulses (0.05 Hz) were applied from aholding potential of −80 mV to +20 mV for 2 seconds and tail currentswere elicited following the test pulses by stepping the voltage to −65mV. The effects from the compound were calculated by measuringinhibition of peak tail current

Sodium Channel Patch Clamp

Whole-cell patch-clamp was used to directly measure inward sodiumcurrents in HEK-293 cells expressing the human cardiac sodium channel,SCN5A. The compound was tested at a protein-free aqueous buffer. Fordetermining steady state inhibition, sodium currents were elicited every5 seconds using the following voltage protocol: cells were held at apotential of −90 mV and stepped to −20 mV for 60 ms. Effects werecalculated by measuring inhibition of peak current during the test pulseto −20 mV. Rate-dependence of inhibition was assessed by stimulation atfrequencies of 1 Hz and 4 Hz.

Single-Dose Pharmacokinetics in Rats

Male Sprague-Dawley rats (250-300 g) were used for the pharmacokineticstudies. Rats were fasted overnight prior to PO dosing and fed 4 h postdose. Blood samples (˜0.3 mL) were collected from the jugular vein intoK₂EDTA-containing tubes and then centrifuged at 4° C. (1500-2000×g) toobtain plasma. In an oral bioavailability study, 2 groups of animals(N=2-3 per group) received the test compound either as an intravenous(IV) infusion (over 10 min) via the jugular vein or by oral gavage.Serial blood samples were obtained at 0.17 (for IV only), 0.25, 0.5,0.75, 1, 2, 4, 6, 8, and 24 h post dose. Plasma samples, obtained bycentrifugation at 4° C. (1500-2000×g), were stored at −20° C. untilanalysis by LC/MS/MS.

Single-Dose Pharmacokinetics in Monkeys

The pharmacokinetics of various test compounds were evaluated in malecynomolgus monkeys in a crossover-design. Monkeys were fasted overnightprior to PO dosing and fed 4 h post dose. A group of 1-3 animals (3 to 5kg) received the compound by IV infusion (over 10 min) via a femoralvein and by oral gavage, with a 1-week washout between treatments.Serial blood samples (˜0.3 mL) were collected from a femoral artery at0.17 (IV only), 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h post dose, andcentrifuged at 4° C. (1500-2000×g) to obtain plasma. Samples were storedat −20° C. until analysis by LC/MS/MS.

Data Analysis for Pharmacokinetic Assays

The pharmacokinetic parameters were obtained by non-compartmentalanalysis of plasma concentration vs. time data (Kinetica software,Version 4.2, InnaPhase Corporation, Philadelphia, Pa.). The peakconcentration (C_(max)) and time for C_(max) were recorded directly fromexperimental observations. The area under the curve from time zero tothe last sampling time (AUC(0-T)) was calculated using a combination oflinear and log trapezoidal summations. The total plasma clearance(CLTp), steady-state volume of distribution (Vss), apparent eliminationhalf-life (T1/2) and mean residence time (MRT) were estimated after IVadministration. Estimations of T1/2 was made using a minimum of 3 timepoints with quantifiable concentrations. The absolute oralbioavailability (F) was estimated as the ratio of dose-normalized AUCvalues following oral and IV doses.

CCR-2 Calcium Mobilization

Human CCR-2-mediated intracellular calcium flux assay was establishedwith the human monocytic cell line THP-1 monocytic cell line. THP-1cells were first loaded with fluorophore by resuspending them in aglucose- and HEPES-buffered PBS (pH 7.4) containing 4 μM fluo-3(Molecular Probes) and 1.25 mM Probenecid and then incubated for 60minutes at 37° C. After washing once to remove excess fluo-3, the cellswere re-suspended in washing buffer (containing phenol red-free RPMI)with 1.25 mM Probenecid, and plated into 96-well plate at 2×10⁵/well.The plate was placed in a FLIPR®-1 (Molecular Devices) that uses anargon-ion laser to excite the cells and robotically adds the testcompound and human MCP-1 while monitoring changes in fluorescence. Testcompound dilutions with a range of concentration from 0 to 100 nM orbuffer alone were added to each well, centrifuged and incubated for 10minutes. Recombinant human MCP-1 (PeproTech Inc.) was then added to afinal concentration of 10 nM. The fluorescence shift was monitored andthe base-to-peak excursion computed automatically. All conditions weretested in duplicate. The inhibition achieved by graded concentrations ofcompound was calculated as a percentage of the compound-free MCP-1control.

CCR-5 Calcium Mobilization

A similar procedure to the CCR-2 calcium mobilization set forth abovewas adopted except that MIP-1β (50 nM) was the ligand and the cell linewas HT1080/CCR-5 in which endogenous CCR-5 is upregulated by randomactivation of gene expression (RAGE) technology.

CCR-2 GTP-γS Exchange

The MCP-1 dependent binding of [³⁵S]-GTPγS to CCR-2 was determined usingmembranes prepared from the HT1080 human cell line in which endogenousCCR-2 was upregulated by RAGE technology (Athersys). Each reaction (200μL) contained 20 mM Na-HEPES, 10 mM MgCl₂, 50 mM NaCl, 0.1% BSA (Sigma),1% DMSO, and 10 μM GDP (pH 7.4). The EC50 for MCP-1 dependent binding of[³⁵S]-GTPγS was determined by varying the MCP-1 concentrations from 1 pMto 1 μM. Reactions were incubated for 90 minutes hour at roomtemperature and [³⁵S]-GTPγS/Gα_(i) complexes were collected on aMillipore MAFC 96 well filter plate. The inhibition of MCP-1 dependent[³⁵S]-GTPγS binding to CCR-2 membranes by the test compound wasdetermined at 1 nM MCP-1 under identical conditions. Data were analyzedusing the ligand binding software from Graphpad Prism 4.

CCR-5 GTP-γS Exchange

A similar procedure to the CCR-2 GTP-γS exchange procedure set forthabove was adopted except that MIP-1α/LD78β was the ligand and the cellline was CCR-5/HT1080. MIP-1α/LD78β was used as the CCR-5 ligand becauseit provided a bigger signal to noise ratio than MIP-1β.

CCR-2 Whole Blood Integrin (CD11b) Upregulation

A CCR-2-dependent CD11b upregulation assay was established with humanwhole blood. Whole blood (100 μl) was pre-incubated with a concentrationrange of Example 1 at 37° C. for 10 minutes. Human recombinant MCP-1 (10μl of 100 nM) was then added to each reaction to a final concentrationof 100 nM, except for unstimulated control reactions. The reactions wereincubated for 30 minutes at 37° C. After incubation, 1 ml of ice coldFACS (PBS with 10% FBS) buffer was added, and the samples werecentrifuged at 1500 rpm for 5 minutes and re-suspended in 50 μl of FACSbuffer. The cells were then incubated with 20 μl of anti-CD14-FITC/anti-CD11b-PE solution for 20 minutes on ice in the darkfollowed by addition of 1 ml of 1×FACS lysing solution (BectonDickinson) to each reaction. The samples were then incubated for 30minutes on ice in the dark. Following fixation and red blood cell lysis,the cells were centrifuged and resuspended in 200 μl FACS-lysingsolution. Samples were analyzed by flow cytometry within 1 hour ofstaining using a FACSCalibur flow cytometer. Data acquisition andanalysis were performed using CellQuestPro software. A sequential gatingstrategy was used to analyze the CD14^(high) CD11b⁺ monocyte population.For analysis, CD11b was measured as median fluorescence intensity (MFI).

CCR-5 Whole Blood CD11b Upregulation

Similar procedure to the CCR-2 whole blood CD11b upregulation procedureset forth above was adopted except that MIP-13 (50 nM) was used as theligand.

Find below data for compared compounds (See WO 2008/014381 A1, WO2008/014360 A1 and WO 2008/014361 A1). The comparative data shows theunexpected combination of equipotent dual CCR-2 and CCR-5 receptorinhibitory and desirable pharmacological characteristics.

TABLE 2 Comparative In vitro Data CCR-2 CCR-5 hERG Na⁺ channel PAMPABinding Binding FLUX binding permeability Compound IC₅₀ (nM) IC₅₀ (nM)IC₅₀ (nM) (% inhibition) (nm/sec) Example 12as 0.27 (1) Not 2,800 NotNot WO 2005/021500 available available available Example 12aj 0.43 ±0.06 (2)  Not 770 Not Not WO 2005/021500 available available availableExample 2k 0.7 ± 0.3 (23) 2.3 ± 1.8 51,000 97%, 10,000 nM 529 ± 157 (9)WO 2005/021500 Example 12bd 1.15 ± 0.07 (2)  Not >80,000 54%, 10,000 nM392 WO 2005/021500 available Example 8a 1.83 ± 0.80 (12) Not >80,000 3%,10,000 nM  94 ± 58 (10) WO 2005/021500 available 33%, 30,000 nM Example8e 2.20 ± 0.03 (2)  Not >80,000 6%, 10,000 nM 2 ± 2 (2) WO 2005/021500available Example 9c 0.96 ± 0.26 (19) Not >80,000 48%, 10,000 nM 145 ±71 (8)  WO 2005/021500 available 75%, 30,000 nM Example 1 1.4 ± 0.5 (18)23.6 ± 12.0 >80,000 0%, 10,000 nM; 443 ± 114 (8) WO 2008/014381 21%,30,000 nM Example 1 2.74 ± 1.34 (15) 6.3 ± 1.5 >80,000 13%, 10,000 nM560 ± 86 (5)  WO 2008/014360 32%, 30,000 nM Example 1 6.2 ± 2.7   3.6 ±1.8 >80,000 46%, 30,000 nM 336 Present Invention

TABLE 3 Additional Comparative In vitro Data CCR-2 CCR-5 hERG patch Na⁺channel Chemotaxis Chemotaxis clamp patch clamp Compound IC₅₀ (nM) IC₅₀(nM) (% Inhib.) (% Inhib.) Example 2k 0.24 ± 0.16 (12) Not 83%, 10,000nM 52%, 10,000 nM U.S. Pat. No. available 90%, 30,000 nM 7163937 Example8a 2.63 ± 1.24 (4)  Not 4%, 10,000 nM 22%, 10,000 nM WO 2005/021500available 49%, 30,000 nM Example 9c 0.21 Not 4%, 10,000 nM 19%, 10,000nM WO 2005/021500 available 39%, 30,000 nM Example 1 0.71 ± 0.16 (22)Not 33%, 10,000 nM 17%, 10,000 nM WO 2008/014381 available 61%, 30,000nM 19%, 30,000 nM Example 1 0.8 ± 0.5 (16) 1.1 ± 0.7 12%, 10,000 nM 29%,30,000 nM WO 2008/014360 19%, 30,000 nM Example 1 0.8 ± 0.8   1.1 ± 0.61.2%, 10,000 nM 15-20%, 10,000 nM Present Invention 9.2%, 30,000 nM13-16%, 30,000 nM

TABLE 4 Additional Comparative In vitro Data CCR-2 Ca²⁺ CCR-5²⁺ CCR-2CCR-5 Flux Flux GTP-γS GTP-γS Compound IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM)IC₅₀ (nM) Example 2k Not Not Not Not U.S. Pat. No. available availableavailable available 7163937 Example 1 0.9 24.0 5 111.0 WO 2008/014381Example 1 0.8 ± 0.6 5.9 ± 3.3 3.5 ± 1.9 5.4 ± 3.2 WO 2008/014360 Example1 2.9 ± 1.5 2.0 ± 1.4 3.5 ± 1.9 6.1 ± 1.2 Present Invention

TABLE 5 Additional Comparative In vitro Data CCR-2 CD11b CCR-5 CD11bCompound IC₅₀ (nM) IC₅₀ (nM) Example 2k 4.7 ± 0.9 4.3 ± 4.4 U.S. Pat.No. 7,163,937 Example 1 0.77 ± 0.40 Not WO 2008/014381 available Example1 2.6 ± 2.2 34.7 ± 11.0 WO 2008/014360 Example 1 4.8 5.7 PresentInvention

Comparative In vivo Pharmacokinetic Data in the Rat Dose IV/PO Cl OralAUC Compound (mg/kg) (mL/min/kg) F % (nM*h) Example 2k 2.5/25   40 689294 WO 2005/021500 Example 8a 6/72 42 1.4 690 WO 2005/021500 Example 9c4/43 54 14 1855 WO 2005/021500 Example 1 2/10 43 51 3794 WO 2008/014381Example 1 2/10 25 79 10169 WO 2008/014360 Example 1 2/10 49.7 94 6500Present Invention

TABLE 7 Comparative In vivo Pharmacokinetic Data in the Monkey DoseIV/PO Cl Oral AUC Compound (mg/kg) (mL/min/kg) F % (nM*h) Example 2k 1/1.4 25 46 862 WO 2005/021500 Example 8a 1/11 14 9.4 1896 WO2005/021500 Example 9c 1/10 12 26 6763 WO 2005/021500 Example 1  1/1.323 47 836 WO 2008/014381 Example 1 1/1  12 95 2352 WO 2008/014360Example 1 1/1  16.4 63 1300 Present Invention

Surprisingly, it was discovered that Example 1 of the present inventionare not predominantly active against CCR-2 or CCR-5, but instead areequipotent dual antagonists, as measured by their CCR-2 and CCR-5binding ability, and posses beneficial pharmacological characteristics.See Tables 2 to 5. For example, see Table wherein Example 1 isequipotent against CCR-2 and CCR-5 while Examples 1 of WO 2008/014381and WO 2008/014360 are predominantly active against CCR-2.

UTILITY

The compounds of the examples are shown to be modulators of chemokinereceptor activity using assays know by those skilled in the art. In thissection, we describe such assays and give their literature reference.More assays are described herein in the section titled “ComparativePharmacological Characteristics”, supra. By displaying activity in theseassays of MCP-1 antagonism, compounds of the examples are expected to beuseful in the treatment of human diseases associated with chemokines andtheir cognate receptors. The definition of activity in these assays is acompound demonstrating an IC₅₀ of 30 μM or lower in concentration whenmeasured in a particular assay.

Antagonism of MCP-1-Induced Calcium Influx

(Sullivan et al., Methods Mol. Biol., 114:125-133 (1999))

At least one compounds described in the examples have activity in theantagonism of MCP-1-induced calcium influx assay described here.

Calcium mobilization is measured using the fluorescent Ca²⁺ indicatordye, Fluo-3. Cells are incubated at 8×10⁵ cells/ml in phosphate-bufferedsaline containing 0.1% bovine serum albumin, 20 mM HEPES buffer, 5 mMglucose, 1% fetal bovine serum, 4 μM Fluo-3 AM and 2.5 mM Probenecid for60 minutes at 37° C. Cells used for such calcium assays can includehuman monocytes isolated as described by Weiner et al., J. Immunol.Methods, 36:89-97 (1980) or cell lines which expresses the endogenousCCR-2 receptor such as THP-1 and MonoMac-6. The cells are then washedthree times in phosphate-buffered saline containing 0.1% bovine serumalbumin, 20 mM HEPES, 5 mM glucose and 2.5 mM Probenecid. The cells areresuspended in phosphate-buffered saline containing 0.5% bovine serumalbumin, 20 mM HEPES and 2.5 mM Probenecid at a final concentration of2−4×10⁶ cells/ml. Cells are plated into 96-well, black-wall microplates(100 μl/well) and the plates centrifuged at 200×g for 5 minutes. Variousconcentrations of compound are added to the wells (50 μl/well) and after5 minutes, 501/well of MCP-1 is added to give a final concentration of10 nM. Calcium mobilization is detected by using a fluorescent-imagingplate reader. The cell monolayer is excited with an argon laser (488 nM)and cell-associated fluorescence measured for 3 minutes, (every secondfor the first 90 seconds and every 10 seconds for the next 90 seconds).Data are generated as arbitrary fluorescence units and the change influorescence for each well determined as the maximum-minimumdifferential. Compound-dependent inhibition is calculated relative tothe response of MCP-1 alone.

Mammalian chemokine receptors provide a target for interfering with orpromoting immune cell function in a mammal, such as a human. Compoundsthat inhibit or promote chemokine receptor function are particularlyuseful for modulating immune cell function for therapeutic purposes.Accordingly, the present invention is directed to compounds which areuseful in the prevention and/or treatment of a wide variety ofinflammatory, infectious, and immunoregulatory disorders and diseases,including asthma and allergic diseases, infection by pathogenic microbes(which, by definition, includes viruses), as well as autoimmunepathologies such as the rheumatoid arthritis and atherosclerosis.

For example, an instant compound which inhibits one or more functions ofa mammalian chemokine receptor (e.g., a human chemokine receptor) may beadministered to inhibit (i.e., reduce or prevent) inflammation orinfectious disease. As a result, one or more inflammatory process, suchas leukocyte emigration, adhesion, chemotaxis, exocytosis (e.g., ofenzymes, histamine) or inflammatory mediator release, is inhibited.

Similarly, an instant compound which promotes one or more functions ofthe mammalian chemokine receptor (e.g., a human chemokine) asadministered to stimulate (induce or enhance) an immune or inflammatoryresponse, such as leukocyte emigration, adhesion, chemotaxis, exocytosis(e.g., of enzymes, histamine) or inflammatory mediator release,resulting in the beneficial stimulation of inflammatory processes. Forexample, eosinophils can be recruited to combat parasitic infections. Inaddition, treatment of the aforementioned inflammatory, allergic andautoimmune diseases can also be contemplated for an instant compoundwhich promotes one or more functions of the mammalian chemokine receptorif one contemplates the delivery of sufficient compound to cause theloss of receptor expression on cells through the induction of chemokinereceptor internalization or the delivery of compound in a manner thatresults in the misdirection of the migration of cells.

In addition to primates, such as humans, a variety of other mammals canbe treated according to the method of the present invention. Forinstance, mammals, including but not limited to, cows, sheep, goats,horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine,canine, feline, rodent or murine species can be treated. However, themethod can also be practiced in other species, such as avian species.The subject treated in the methods above is a mammal, male or female, inwhom modulation of chemokine receptor activity is desired. “Modulation”as used herein is intended to encompass antagonism, agonism, partialantagonism and/or partial agonism.

Fluorometric Imaging Plate Reader (FLIPR®)-Based Functional Assay

HT1080 cells (clone 3559.1.6) were plated at 10,000 cells/well (30microliters) in 384-well plates (black/clear bottom BIOCOAT® PDL,Beckton Dickinson) and charged with 30 microliters/well of Fluo-4 AMfluorescent dye (prepared by dissolving 1 mg Fluo-4 AM in 440microliters DMSO and diluting with 100 microliters of pluronic solutionbefore diluting further with 10 mL of Hanks buffer). The cells wereincubated at 37° C. with 5% CO₂ for 30 min before being washed threetimes and suspended in Assay Buffer (20 mM HEPES, 1.2 mM CaCl₂, 5 mMMgCl₂, 2.5 mM Probenecid, 0.5% BSA, 1× Hanks). The test article wasserially diluted in DMSO and then diluted 1:10 with Assay Buffer beforebeing added to the cells (10 microliters/well). Using FLIPR®, the plateswere read (10-70 sec) for induction of flux (i.e., agonist activity).The cells were then further charged with Agonist Solution (30microliters/well; prepared by diluting 30 microliters of 100 microMolarMIP-1 beta in 100 mL of Assay Buffer; this protocol delivers a finalconcentration of 5 nM MIP-1 beta in the assay) and the plates were readusing FLIPR® for one minute. Antagonist activity of the test article wasdetermined relative to 0.4% DMSO/Buffer negative control.

In Vivo Assay(s) and Efficacy

N-((1R,2S,5R)-5-(tert-Butylamino)-2-((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide(also referred to as “Example 1”) was evaluated in the vivo assaydescribed below.

48-Hour Thioglycollate (TG)-Induced Peritonitis Model in hCCR-2 KI MouseMethods

The hCCR-2 KI mice (C57BL/6-SVJ129) were injected intraperitoneally with1 ml of thioglycollate (TG) (Hardy Diagnostics). For each study, eightmale mice per group were used. Example 1 was dosed orally 1 hour priorto TG injection. The vehicle used was 0.01 N HCl in water. Forty-eighthours post TG injection, peritoneal lavages were performed by injecting5 ml PBS/10 mM EDTA/10% BSA into the peritoneal cavity.

For the 48-hour TG peritonitis study, Example 1 was dosed twice a daywith the first dose one hour prior to TG injection. Total peritonealcell counts were obtained on isolated cells by a cell counter. Cytospinswere performed to determine differential leukocyte counts. The cellswere stained for 3 minutes with Wright-Giemsa Stain (Sigma-Aldrich) andthen rinsed with deionized water for 5 minutes. Differential counts werecalculated based on a total of 200 cells counted per sample. Blood wasalso collected from the retro-orbital sinus at the end of each study inEDTA for determination of drug concentration.

For flow cytometric analysis, peritoneal exudate cells (1×10⁶) werewashed once with FACS buffer (PBS/0.5% BSA) and resuspended in FACSbuffer. Cells were incubated with an Fc-blocking antibody (BDPharmingen) on ice for 15 min followed by addition of the followingantibodies (BD Pharmingen): PE conjugated anti-F4/80, FITC conjugatedanti-Ly6C, and Alexa 647 conjugated anti-hCCR-2. After 45 min on ice,cells were fixed by BD CYTOFIX® for 15 min on ice, washed twice withFACS buffer, and resuspended in 200 μl FACS buffer. Cellular events(40,000) were acquired for each sample and data were analyzed usingFloJo software (TreeStar). A FSC/SSC gate was set to include allmonocytes (low SSC, higher FSC) while excluding granulocytes from theanalysis. This gated population was then analyzed for Ly6C (FITC), F4/80(PE) expression. Peritoneal monocytes/macrophage numbers were determinedby multiplying total peritoneal cell counts obtained by the cell counterand the percentage of monocytes/macrophages identified by F4/80⁺ cellsfrom flow cytometry. Statistical significance of differences betweenmeans was analyzed using the paired two-tailed t test with significanceset at p values below 0.05.

Results

Example 1 was evaluated in the hCCR-2 KI mouse TG peritonitis model todetermine its EC50 in inhibiting monocyte/macrophage infiltration. Micewere administered thioglycollate, and dosed orally with Example 1 at 10,50, or 160 mg/kg BID. Forty eight hours post TG treatment, peritoneallavage was obtained for cellular infiltrate analysis by flow cytometry.

A dose-dependent inhibition in monocyte/macrophage infiltration wasobserved (FIG. 4). Doses of 10, 50, and 160 mg/kg gave an inhibition of25%, 54% and 63%, respectively. Of the four separate studies withmultiple doses, the maximal inhibition reached was ˜70% and the averageEC50 for inhibition of monocyte/macrophage infiltration by this analysiswas estimated to be 4.9 nM, which correlates well with the in vitro IC50(5.8±2.3 nM) for Example 1 inhibition of ¹²⁵I-mouse MCP-1 binding tohuman CCR-2-expressing cells (hPBMCs).

To assess the in vivo level of receptor occupancy by Example 1 in the48-hour thioglycolate peritonitis model in the hCCR-2 KI mouse, plasmalevels of both Example 1 and mouse MCP-1 were measured. The caveat forthis estimation is that only CCR-2 and its major ligand MCP-1 were takeninto consideration. The receptor occupancy of a ligand in the presenceof a competitive inhibitor is defined by the Gaddum equation:

$\frac{\lbrack{RL}\rbrack}{\left\lbrack R \right\}} = \frac{1}{1 + {\left( {K_{d}/\lbrack L\rbrack} \right)\left( {1 + {\lbrack I\rbrack/K_{i}}} \right)}}$

Since Example 1 is a competitive inhibitor of MCP-1 binding to CCR-2,the amounts of both mouse MCP-1/CCR-2 receptor complex and Example1/CCR-2 receptor complex can be determined using the serum levels ofboth mouse MCP-1 and protein-unbound Example 1 in plasma. The K_(d) formouse MCP-1 binding to hCCR-2 is 0.91+/−0.08 nM (n=8) which wasdetermined in cold competition ligand binding experiments using¹²⁵I-human MCP-1. The average K_(i) for Example 1 binding to hCCR-2 is2.0 nM. The fraction of mouse MCP-1/CCR-2 receptor complexes isdetermined using the form of the equation described above. To determinethe fraction of Example 1/CCR-2 complexes the equation is re-defined as:

$\frac{\lbrack{RI}\rbrack}{\left\lbrack R \right\}} = \frac{1}{1 + {\left( {K_{i}/\lbrack I\rbrack} \right)\left( {1 + {\lbrack L\rbrack/K_{d}}} \right)}}$Finally, the amount of CCR-2 free is determined from:[CCR-2]_(total)=[CCR-2]_(free)+[mouse MCP-1/CCR-2]+[Example 1/CCR-2]

As shown in Table 8, the percent inhibition of monocyte/macrophageinfiltration into the peritoneum at 48 hour reflects the percentage ofExample 1/CCR-2 receptor complex.

TABLE 8 Determination of In vivo Receptor Occupancy of Example 1 inBlood of hCCR-2 KI Mice in the 48-hour TG Peritonitis ModelConcentration Concentration of % inhibition of Mouse free Example 1 inof monocyte/ Dose MCP-1 in plasma (nM) (fold % mouse MCP-1 % Example 1 %free macrophage mg/kg plasma (nM) IC90 CCR-2 binding) bound CCR-2 boundCCR-2 CCR-2 infiltration^(a) 200^(a ) 0.068 136.4 0.11 98.45 1.44 71160  0.046 62.0 0.16 96.72 3.12 63 50 0.048 16.6 0.56 88.74 10.69 54 100.021 1.9 1.17 48.15 50.68 25  0 0.022 0.0 2.36 0.00 97.64 0 ^(a)dosewas selected from a different study from other doses in the table toprovide an example of maximal inhibition. This dose provided a freeplasma concentration of 1.9-fold IC90 the hCCR-2 KI binding.

Collectively, these results clearly demonstrate that Example 1 is apotent blocker of monocyte/macrophage infiltration with an EC50 of ˜4.9nM. Maximal inhibition of monocyte/macrophage infiltration by Example 1can be achieved by 98.5% CCR-2 occupancy with this compound. Notably,the studies have demonstrated a similar reduction (˜70-80%) inmonocyte/macrophage infiltration in CCR-2-deficient mice.

Diseases or conditions of human or other species which can be treatedwith inhibitors of chemokine receptor function, include, but are notlimited to: inflammatory or allergic diseases and conditions, includingrespiratory allergic diseases such as asthma, allergic rhinitis,hypersensitivity lung diseases, hypersensitivity pneumonitis,eosinophilic cellulitis (e.g., Well's syndrome), eosinophilic pneumonias(e.g., Loeffler's syndrome, chronic eosinophilic pneumonia),eosinophilic fasciitis (e.g., Shulman's syndrome), delayed-typehypersensitivity, interstitial lung diseases (ILD) (e.g., idiopathicpulmonary fibrosis, or ILD associated with rheumatoid arthritis,systemic lupus erythematosus, ankylosing spondylitis, systemicsclerosis, Sjögren's syndrome, polymyositis or dermatomyositis);systemic anaphylaxis or hypersensitivity responses, drug allergies(e.g., to penicillin, cephalosporins), eosinophilia-myalgia syndrome dueto the ingestion of contaminated tryptophan, insect sting allergies;autoimmune diseases, such as rheumatoid arthritis, psoriatic arthritis,multiple sclerosis, systemic lupus erythematosus, myasthenia gravis,juvenile onset diabetes; glomerulonephritis, autoimmune thyroiditis,Behcet's disease; graft rejection (e.g., in transplantation), includingallograft rejection or graft-versus-host disease; inflammatory boweldiseases, such as Crohn's disease and ulcerative colitis;spondyloarthropathies; scleroderma; psoriasis (including T-cell mediatedpsoriasis) and inflammatory dermatoses such as an dermatitis, eczema,atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis(e.g., necrotizing, cutaneous, and hypersensitivity vasculitis);eosinophilic myositis, eosinophilic fasciitis; cancers with leukocyteinfiltration of the skin or organs. Other diseases or conditions inwhich undesirable inflammatory responses are to be inhibited can betreated, including, but not limited to, vasculitis, vulnerable plaques,venous neointimal hyperplasia reperfusion injury, dialysis-graftneointimal hyperplasia, arterio-venous shunt intimal hyperplasia,atherosclerosis, certain hematologic malignancies, cytokine-inducedtoxicity (e.g., septic shock, endotoxic shock), polymyositis,dermatomyositis. Infectious diseases or conditions of human or otherspecies which can be treated with inhibitors of chemokine receptorfunction, include, but are not limited to, HIV.

Diseases or conditions of humans or other species which can be treatedwith promoters of chemokine receptor function, include, but are notlimited to: immunosuppression, such as that in individuals withimmunodeficiency syndromes such as AIDS or other viral infections,individuals undergoing radiation therapy, chemotherapy, therapy forautoimmune disease or drug therapy (e.g., corticosteroid therapy), whichcauses immunosuppression; immunosuppression due to congenital deficiencyin receptor function or other causes; and infections diseases, such asparasitic diseases, including, but not limited to helminth infections,such as nematodes (round worms); (Trichuriasis, Enterobiasis,Ascariasis, Hookworm, Strongyloidiasis, Trichinosis, filariasis);trematodes (flukes) (Schistosomiasis, Clonorchiasis), cestodes (tapeworms) (Echinococcosis, Taeniasis saginata, Cysticercosis); visceralworms, visceral larva migraines (e.g., Toxocara), eosinophilicgastroenteritis (e.g., Anisaki sp., Phocanema sp.), cutaneous larvamigraines (Ancylostona braziliense, Ancylostoma caninum). The compoundsof the present invention are accordingly useful in the prevention andtreatment of a wide variety of inflammatory, infectious andimmunoregulatory disorders and diseases.

In addition, treatment of the aforementioned inflammatory, allergic andautoimmune diseases can also be contemplated for promoters of chemokinereceptor function if one contemplates the delivery of sufficientcompound to cause the loss of receptor expression on cells through theinduction of chemokine receptor internalization or delivery of compoundin a manner that results in the misdirection of the migration of cells.

In another aspect, the instant invention may be used to evaluate theputative specific agonists or antagonists of a G protein coupledreceptor. The present invention is directed to the use of thesecompounds in the preparation and execution of screening assays forcompounds that modulate the activity of chemokine receptors.Furthermore, the compounds of this invention are useful in establishingor determining the binding site of other compounds to chemokinereceptors, e.g., by competitive inhibition or as a reference in an assayto compare its known activity to a compound with an unknown activity.When developing new assays or protocols, compounds according to thepresent invention could be used to test their effectiveness.Specifically, such compounds may be provided in a commercial kit, forexample, for use in pharmaceutical research involving the aforementioneddiseases. The compounds of the instant invention are also useful for theevaluation of putative specific modulators of the chemokine receptors.In addition, one could utilize compounds of this invention to examinethe specificity of G protein coupled receptors that are not thought tobe chemokine receptors, either by serving as examples of compounds whichdo not bind or as structural variants of compounds active on thesereceptors which may help define specific sites of interaction.

Compounds disclosed herein are useful to treat or prevent disordersselected from rheumatoid arthritis, osteoarthritis, septic shock,atherosclerosis, aneurism, fever, cardiovascular effects, haemodynamicshock, sepsis syndrome, post ischemic reperfusion injury, malaria,Crohn's disease, inflammatory bowel diseases, mycobacterial infection,meningitis, psoriasis, congestive heart failure, fibrotic diseases,cachexia, graft rejection, autoimmune diseases, skin inflammatorydiseases, multiple sclerosis, radiation damage, hyperoxic alveolarinjury, HIV, HIV dementia, non-insulin dependent diabetes mellitus,asthma, allergic rhinitis, atopic dermatitis, idiopathic pulmonaryfibrosis, bullous pemphigoid, helminthic parasitic infections, allergiccolitis, eczema, conjunctivitis, transplantation, familial eosinophilia,eosinophilic cellulitis, eosinophilic pneumonias, eosinophilicfasciitis, eosinophilic gastroenteritis, drug induced eosinophilia,cystic fibrosis, Churg-Strauss syndrome, lymphoma, Hodgkin's disease,colonic carcinoma, Felty's syndrome, sarcoidosis, uveitis, Alzheimer,Glomerulonephritis, and systemic lupus erythematosus, esophagealsquamous cell carcinoma, neuropathic pain, and obesity.

In another aspect, the compounds are useful to treat or preventinflammatory disorders selected from rheumatoid arthritis,osteoarthritis, atherosclerosis, aneurism, fever, cardiovasculareffects, Crohn's disease, inflammatory bowel diseases, psoriasis,congestive heart failure, multiple sclerosis, autoimmune diseases, skininflammatory diseases.

In another aspect, the compounds are used to treat or preventinflammatory disorders selected from rheumatoid arthritis,osteoarthritis, atherosclerosis, Crohn's disease, inflammatory boweldiseases, and multiple sclerosis.

In another aspect, examples disclosed herein may be useful in for thetreatment of a variety of cancers, including, but not limited to, thefollowing:

carcinoma including that of the bladder (including accelerated andmetastatic bladder cancer), breast, colon (including colorectal cancer),kidney, liver, lung (including small and non-small cell lung cancer andlung adenocarcinoma), ovary, prostate, testes, genitourinary tract,lymphatic system, rectum, larynx, pancreas (including exocrinepancreatic carcinoma), esophagus, stomach, gall bladder, cervix,thyroid, and skin (including squamous cell carcinoma);

hematopoietic tumors of lymphoid lineage including leukemia, acutelymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma,T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy celllymphoma, histiocytic lymphoma, and Burketts lymphoma;

hematopoietic tumors of myeloid lineage including acute and chronicmyelogenous leukemias, myelodysplastic syndrome, myeloid leukemia, andpromyelocytic leukemia;

tumors of the central and peripheral nervous system includingastrocytoma, neuroblastoma, glioma, and schwannomas;

tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma,and osteosarcoma; and

other tumors including melanoma, xenoderma pigmentosum,keratoactanthoma, seminoma, thyroid follicular cancer, andteratocarcinoma.

In another embodiment, disclosed herein are methods of treating cancer,wherein the cancer is selected from breast cancer, liver cancer,prostate cancer, and melanoma. Additionally, compounds disclosed hereinmay be useful in the treatment of ovarian cancer, and multiple myeloma.

The present invention provides methods for the treatment of a variety ofnon-cancerous proliferative diseases.

Combined therapy to prevent and treat inflammatory, infectious andimmunoregulatory disorders and diseases, including asthma and allergicdiseases, as well as autoimmune pathologies such as rheumatoid arthritisand atherosclerosis, and those pathologies noted above is illustrated bythe combination of the compounds of this invention and other compoundswhich are known for such utilities. For example, in the treatment orprevention of inflammation, the present compounds may be used inconjunction with an anti-inflammatory or analgesic agent such as anopiate agonist, a lipoxygenase inhibitor, a cyclooxygenase-2 inhibitor,an interleukin inhibitor, such as an interleukin-1 inhibitor, a tumornecrosis factor inhibitor, an NMDA antagonist, an inhibitor or nitricoxide or an inhibitor of the synthesis of nitric oxide, a non-steroidalanti-inflammatory agent, a phosphodiesterase inhibitor, or acytokine-suppressing anti-inflammatory agent, for example with acompound such as acetaminophen, aspirin, codeine, fentanyl, ibuprofen,indomethacin, ketorolac, morphine, naproxen, phenacetin, piroxicam, asteroidal analgesic, sufentanyl, sunlindac, interferon alpha and thelike. Similarly, the instant compounds may be administered with a painreliever; a potentiator such as caffeine, an H2-antagonist, simethicone,aluminum or magnesium hydroxide; a decongestant such as phenylephrine,phenylpropanolamine, pseudophedrine, oxymetazoline, ephinephrine,naphazoline, xylometazoline, propylhexedrine, or levodesoxy-ephedrine;and antitussive such as codeine, hydrocodone, caramiphen,carbetapentane, or dextramethorphan; a diuretic; and a sedating ornon-sedating antihistamine. Likewise, compounds disclosed herein may beused in combination with other drugs that are used in thetreatment/prevention/suppression or amelioration of the diseases orconditions for which compound of the present invention are useful. Suchother drugs may be administered, by a route and in an amount commonlyused therefore, contemporaneously or sequentially with a compound of thepresent invention. When a compound is used contemporaneously with one ormore other drugs, a pharmaceutical composition containing such otherdrugs in addition to the compound of the present invention may be used.Accordingly, the pharmaceutical compositions include those that alsocontain one or more other active ingredients, in addition to a compoundof the present disclosure.

Examples of other active ingredients that may be combined with acompound of the present invention, either administered separately or inthe same pharmaceutical compositions, include, but are not limited to:(a) integrin antagonists such as those for selectins, ICAMs and VLA-4;(b) steroids such as beclomethasone, methylprednisolone, betamethasone,prednisone, dexamethasone, and hydrocortisone; (c) immunosuppressantssuch as cyclosporin, tacrolimus, rapamycin and other FK-506 typeimmunosuppressants; (d) antihistamines (H1-histamine antagonists) suchas bromopheniramine, chlorpheniramine, dexchlorpheniramine,triprolidine, clemastine, diphenhydramine, diphenylpyraline,tripelennamine, hydroxyzine, methdilazine, promethazine, trimeprazine,azatadine, cyproheptadine, antazoline, pheniramine pyrilamine,astemizole, terfenadine, loratadine, cetirizine, fexofenadine,descarboethoxyloratadine, and the like; (e) non-steroidalanti-asthmatics such as b2-agonists (terbutaline, metaproterenol,fenoterol, isoetharine, albuterol, bitolterol, and pirbuterol),theophylline, cromolyn sodium, atropine, ipratropium bromide,leukotriene antagonists (zafirlukast, montelukast, pranlukast,iralukast, pobilukast, SKB-102,203), leukotriene biosynthesis inhibitors(zileuton, BAY-1005); (f) non-steroidal antiinflammatory agents (NSAIDs)such as propionic acid derivatives (alminoprofen, benxaprofen, bucloxicacid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen,ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin,pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen),acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac,diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac,isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, andzomepirac), fenamic acid derivatives (flufenamic acid, meclofenamicacid, mefenamic acid, niflumic acid and tolfenamic acid),biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams(isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetylsalicylic acid, sulfasalazine) and the pyrazolones (apazone,bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone);(g) cyclooxygenase-2 (COX-2) inhibitors; (h) inhibitors ofphosphodiesterase type IV (PDE-IV); (i) other antagonists of thechemokine receptors; (j) cholesterol lowering agents such as HMG-CoAreductase inhibitors (lovastatin, simvastatin, pravastatin, fluvastatin,atorvastatin, and other statins), sequestrants (cholestyramine andcolestipol), nicotinic acid, fenofibric acid derivatives (gemfibrozil,clofibrat, fenofibrate and benzafibrate), and probucol; (k)anti-diabetic agents such as insulin, sulfonylureas, biguanides(metformin), a-glucosidase inhibitors (acarbose) and glitazones(troglitazone ad pioglitazone); (l) preparations of interferons(interferon alpha-2a, interferon-2B, interferon alpha-N3, interferonbeta-1a, interferon beta-1b, interferon gamma-1b); (m) antiviralcompounds such as efavirenz, nevirapine, indinavir, ganciclovir,lamivudine, famciclovir, and zalcitabine; (o) other compound such as5-aminosalicylic acid an prodrugs thereof, antimetabolites such asazathioprine and 6-mercaptopurine, and cytotoxic cancer chemotherapeuticagents. The weight ratio of the compound of the present invention to thesecond active ingredient may be varied and will depend upon theeffective doses of each ingredient.

Generally, an effective dose of each will be used. Thus, for example,when a compound is combined with an NSAID the weight ratio of thecompound of the present invention to the NSAID will generally range fromabout 1000:1 to about 1:1000, or alternatively from about 200:1 to about1:200. Combinations of a compound of the present invention and otheractive ingredients will generally also be within the aforementionedrange, but in each case, an effective dose of each active ingredientshould be used.

In treating cancer, a combination of chemotherapeutic agents and/orother treatments (e.g., radiation therapy) is often advantageous. Thesecond (or third) agent may have the same or different mechanism ofaction than the primary therapeutic agent. It may be especially usefulto employ cytotoxic drug combinations wherein the two or more drugsbeing administered act in different manners or in different phased ofthe cell cycle, and/or where the two or more drugs have overlappingtoxicities or side effects, and/or where the drugs being combined eachhas a demonstrated efficacy in treating the particular disease statemanifested by the patient.

Accordingly, compounds disclosed herein (or other formulae disclosedherein) may be administered in combination with other anti-cancer andcytotoxic agents and treatments useful in the treatment of cancer orother proliferative diseases. The invention herein further comprises useof the compounds herein (or other formulae disclosed herein), inpreparing medicaments for the treatment of cancer, and/or it comprisesthe packaging of the compounds of herein together with instructions thatthe compounds be used in combination with other anti-cancer or cytotoxicagents and treatments for the treatment of cancer. The present inventionfurther comprises combinations of the compounds of and one or moreadditional agents in kit form, e.g., where they are packaged together orplaced in separate packages to be sold together as a kit, or where theyare packaged to be formulated together.

The second (or more) anti-cancer agents may be selected from any one ormore of the following:

alkylating agents (including nitrogen mustards, alkyl sulfonates,nitrosoureas, ethylenimine derivatives, and triazenes); anti-angiogenics(including matrix metalloproteinase inhibitors); antimetabolites(including adenosine deaminase inhibitors, folic acid antagonists,purine analogues, and pyrimidine analogues); antibiotics or antibodies(including monoclonal antibodies, CTLA-4 antibodies, anthracyclines);aromatase inhibitors;

cell-cycle response modifiers; enzymes; farnesyl-protein transferaseinhibitors;

hormonal and antihormonal agents and steroids (including syntheticanalogs, glucocorticoids, estrogens/anti-estrogens [e.g., SERMs],androgens/anti-androgens, progestins, progesterone receptor agonists,and luteinizing hormone-releasing [LHRH] agonists and antagonists);insulin-like growth factor (IGF)/insulin-like growth factor receptor(IGFR) system modulators (including IGFR1 inhibitors);integrin-signaling inhibitors; kinase inhibitors (including multi-kinaseinhibitors and/or inhibitors of Src kinase or Src/abl, cyclin dependentkinase [CDK] inhibitors, panHer, Her-1 and Her-2 antibodies, VEGFinhibitors, including anti-VEGF antibodies, EGFR inhibitors,mitogen-activated protein [MAP] inhibitors, MEK inhibitors, Aurorakinase inhibitors, PDGF inhibitors, and other tyrosine kinase inhibitorsor serine/threonine kinase inhibitors;

microtubule-disruptor agents, such as ecteinascidins or their analogsand derivatives; microtubule-stabilizing agents such as taxanes, and thenaturally-occurring epothilones and their synthetic and semi-syntheticanalogs;

microtubule-binding, destabilizing agents (including vinca alkaloids);and

topoisomerase inhibitors; prenyl-protein transferase inhibitors;platinum coordination complexes; signal transduction inhibitors; andother agents used as anti-cancer and cytotoxic agents such as biologicalresponse modifiers, growth factors, and immune modulators.

Additionally, the compounds of the present invention can be formulatedor co-administered with other therapeutic agents that are selected fortheir particular usefulness in addressing side effects associated withthe aforementioned conditions. For example, compounds of the inventionmay be formulated with agents to prevent nausea, hypersensitivity andgastric irritation, such as antiemetics, and H₁ and H₂ antihistaminics.

The above other therapeutic agents, when employed in combination withthe compounds of the present invention, can be used, for example, inthose amounts indicated in the Physicians' Desk Reference (PDR) or asotherwise determined by one of ordinary skill in the art.

The compounds are administered to a mammal in a therapeuticallyeffective amount. By “therapeutically effective amount” it is meant anamount of a compound of the present disclosure that, when administeredalone or in combination with an additional therapeutic agent to amammal, is effective to prevent or ameliorate the disease condition orthe progression of the disease.

Dosage and Formulation

The compounds of this disclosure can be administered in such oral dosageforms as tablets, capsules (each of which includes sustained release ortimed release formulations), pills, powders, granules, elixirs,tinctures, suspensions, syrups, and emulsions. They may also beadministered in intravenous (bolus or infusion), intraperitoneal,subcutaneous, or intramuscular form, all using dosage forms well knownto those of ordinary skill in the pharmaceutical arts. They can beadministered alone, but generally will be administered with apharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

The dosage regimen for the compounds of the present invention will, ofcourse, vary depending upon known factors, such as the pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the species, age, sex, health, medical condition, andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; the route ofadministration, the renal and hepatic function of the patient, and theeffect desired. A physician or veterinarian can determine and prescribethe effective amount of the drug required to prevent, counter, or arrestthe progress of the disorder.

By way of general guidance, the daily oral dosage of each activeingredient, when used for the indicated effects, will range betweenabout 0.001 to 1000 mg/kg of body weight, or between about 0.01 to 100mg/kg of body weight per day, or alternatively, between about 1.0 to 20mg/kg/day. Intravenously, the doses will range from about 1 to about 10mg/kg/minute during a constant rate infusion. Compounds of thisinvention may be administered in a single daily dose, or the total dailydosage may be administered in divided doses of two, three, or four timesdaily. In one embodiment, the daily oral dosage of the active ingredientis between 3 and 600 mg either administered once daily or in divideddoses administered twice daily. Alternatively, the active ingredient maybe administered in doses of 10-20 mg administered twice daily or 40 to100 mg administered once daily. Alternatively, the active ingredient maybe administered a dose of 12.5 mg twice a day or 75 mg once a day.Alternatively, the active ingredient may be administered in doses of 3,10, 30, 100, 300, and 600 mg administered either once or twice a day.

Compounds of this invention can be administered in intranasal form viatopical use of suitable intranasal vehicles, or via transdermal routes,using transdermal skin patches. When administered in the form of atransdermal delivery system, the dosage administration will, of course,be continuous rather than intermittent throughout the dosage regimen.

The compounds are typically administered in admixture with suitablepharmaceutical diluents, excipients, or carriers (collectively referredto herein as pharmaceutical carriers) suitably selected with respect tothe intended form of administration, that is, oral tablets, capsules,elixirs, syrups and the like, and consistent with conventionalpharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic, pharmaceutically acceptable, inert carrier such as lactose,starch, sucrose, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like;for oral administration in liquid form, the oral drug components can becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents, and coloring agents can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucoseor beta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Lubricants used in thesedosage forms include sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum, and the like.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

Compounds of the present invention may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, andcrosslinked or amphipathic block copolymers of hydrogels.

Dosage forms (pharmaceutical compositions) suitable for administrationmay contain from about 1 milligram to about 100 milligrams of activeingredient per dosage unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.5-95% by weight based on the total weight of the composition.

Gelatin capsules may contain the active ingredient and powderedcarriers, such as lactose, starch, cellulose derivatives, magnesiumstearate, stearic acid, and the like. Similar diluents can be used tomake compressed tablets. Both tablets and capsules can be manufacturedas sustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),and related sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration may contain a water soluble saltof the active ingredient, suitable stabilizing agents, and if necessary,buffer substances. Antioxidizing agents such as sodium bisulfite, sodiumsulfite, or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumEDTA. In addition, parenteral solutions can contain preservatives, suchas benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

Representative useful pharmaceutical dosage-forms for administration ofthe compounds of this invention can be illustrated as follows:

Capsules

A large number of unit capsules can be prepared by filling standardtwo-piece hard gelatin capsules each with 100 milligrams of powderedactive ingredient, 150 milligrams of lactose, 50 milligrams ofcellulose, and 6 milligrams magnesium stearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil or olive oil may be prepared and injected by means of apositive displacement pump into gelatin to form soft gelatin capsulescontaining 100 milligrams of the active ingredient. The capsules shouldbe washed and dried.

Tablets

Tablets may be prepared by conventional procedures so that the dosageunit is 100 milligrams of active ingredient, 0.2 milligrams of colloidalsilicon dioxide, milligrams of magnesium stearate, 275 milligrams ofmicrocrystalline cellulose, 11 milligrams of starch and 98.8 milligramsof lactose. Appropriate coatings may be applied to increase palatabilityor delay absorption.

Injectable

A parenteral composition suitable for administration by injection may beprepared by stirring 1.5% by weight of active ingredient in 10% byvolume propylene glycol and water. The solution should be made isotonicwith sodium chloride and sterilized.

Suspension

An aqueous suspension can be prepared for oral administration so thateach 5 mL contain 100 mg of finely divided active ingredient, 200 mg ofsodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g ofsorbitol solution, U.S.P., and 0.025 mL of vanillin.

Where the compounds of this invention are combined with otheranticoagulant agents, for example, a daily dosage may be about 0.1 to100 milligrams of the compound of Formula I and about 1 to 7.5milligrams of the second anticoagulant, per kilogram of patient bodyweight. For a tablet dosage form, the compounds of this inventiongenerally may be present in an amount of about 5 to 10 milligrams perdosage unit, and the second anti-coagulant in an amount of about 1 tomilligrams per dosage unit.

Where two or more of the foregoing second therapeutic agents areadministered with the compound of the examples, generally the amount ofeach component in a typical daily dosage and typical dosage form may bereduced relative to the usual dosage of the agent when administeredalone, in view of the additive or synergistic effect of the therapeuticagents when administered in combination.

Particularly when provided as a single dosage unit, the potential existsfor a chemical interaction between the combined active ingredients. Forthis reason, when the compound of the examples and a second therapeuticagent are combined in a single dosage unit they are formulated such thatalthough the active ingredients are combined in a single dosage unit,the physical contact between the active ingredients is minimized (thatis, reduced). For example, one active ingredient may be enteric coated.By enteric coating one of the active ingredients, it is possible notonly to minimize the contact between the combined active ingredients,but also, it is possible to control the release of one of thesecomponents in the gastrointestinal tract such that one of thesecomponents is not released in the stomach but rather is released in theintestines. One of the active ingredients may also be coated with amaterial which effects a sustained-release throughout thegastrointestinal tract and also serves to minimize physical contactbetween the combined active ingredients. Furthermore, thesustained-released component can be additionally enteric coated suchthat the release of this component occurs only in the intestine. Stillanother approach would involve the formulation of a combination productin which the one component is coated with a sustained and/or entericrelease polymer, and the other component is also coated with a polymersuch as a low viscosity grade of hydroxypropyl methylcellulose (HPMC) orother appropriate materials as known in the art, in order to furtherseparate the active components. The polymer coating serves to form anadditional barrier to interaction with the other component.

These as well as other ways of minimizing contact between the componentsof combination products of the present invention, whether administeredin a single dosage form or administered in separate forms but at thesame time by the same manner, will be readily apparent to those skilledin the art, once armed with the present disclosure.

Additionally, certain compounds disclosed herein may be useful asmetabolites of other compounds. Therefore, in one embodiment, compoundsmay be useful either as a substantially pure compound, which may alsothen be incorporated into a pharmaceutical composition, or may be usefulas metabolite which is generated after administration of the prodrug ofthat compound. In one embodiment, a compound may be useful as ametabolite by being useful for treating disorders as described herein.

“Substantially pure” as used herein is intended to include a compoundhaving a purity greater than about 90 weight percent, including about90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 percent.

As one example, a compound disclosed herein may be substantially pure inhaving a purity greater than about 90 percent (by weight), where theremaining less than about 10 percent of material comprises othermetabolite of the compound, a prodrug of the compound, and/or reactionand/or processing impurities arising from its preparation.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise that as specifically describedherein.

1. A compound having the formula:

or a salt thereof.
 2. The compound of having the formula:


3. The crystalline form of the compound claim 2, wherein the crystallineform is the N-1 Form.
 4. The crystalline form of claim 3, wherein theN-1 Form is characterized by unit cell parameters substantially equal tothe following: Cell dimensions: a=7.3085(6) b=16.257(1) c=22.688(2)α°=90 β°=90 γ°=90 Space group P2₁2₁2₁ Molecules/unit cell (Z): 1Density, calc g-cm⁻³: 1.194 wherein said crystal is at a temperature ofabout −70° C.
 5. The crystalline form as defined in claim 3 ascharacterized by a powder x-ray diffraction pattern substantially inaccordance with that shown in FIG.
 1. 6. The crystalline form as definedin claim 3 which is characterized by a differential scanning calorimetrythermogram substantially in accordance with that shown in FIG. 2, havingan endothermic transition above ca. 205° C.
 7. The crystalline form asdefined in claim 3 which is characterized by a thermal gravimetricanalysis curve in accordance with that shown in FIG.
 3. 8. Apharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable carrier.
 9. The pharmaceutical compositionof claim 8 further comprising at least one additional therapeutic agent.10. A pharmaceutical composition comprising the compound of claim 2 anda pharmaceutically acceptable carrier.
 11. The pharmaceuticalcomposition of claim 10 further comprising at least one additionaltherapeutic agent.
 12. A compound having the formula:

or a salt thereof.
 13. A pharmaceutical composition comprising thecompound of claim 12 and a pharmaceutically acceptable carrier.
 14. Thepharmaceutical composition of claim 13 further comprising at least oneadditional therapeutic agent.
 15. A process for preparingN-((1R,2S,5R)-5-(tert-butylamino)-2((S)-3-(7-tert-butylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamidecomprising the process set forth in the following scheme: